NZ751656B2

NZ751656B2 - Improved thymidine kinase gene

Info

Publication number: NZ751656B2
Application number: NZ751656A
Authority: NZ
Inventors: Robert G Johnson; John P Levy; Joseph Mcnulty; Rebecca A Reed
Original assignee: Genvivo Inc
Priority date: 2013-03-14
Filing date: 2014-03-14
Publication date: 2021-11-02

Abstract

Nucleic acid sequences encoding improved Herpes Simplex Virus Thymidine Kinases (HSV-TK) are provided, including their use in diagnostic and therapeutic applications. The thymidine kinases may be mutated using conservative mutations, non-conservative mutations, or both. In particular, the mutations of the present invention occur at positions 25, 26, 32 or 33 in comparison to the wild-type HSV1-TK. Also provided are gene therapeutic systems, including viral and retroviral particles. of the present invention occur at positions 25, 26, 32 or 33 in comparison to the wild-type HSV1-TK. Also provided are gene therapeutic systems, including viral and retroviral particles.

Description

IMPROVED THYMIDINE KINASE GENE CROSS-REFERENCE This ation is a divisional of New Zealand patent application number 712210, the entire content of which is incorporated into the present specification by this cross-reference.

New Zealand patent application number 712210 is derived from international patent application number which claims the t of U.S. Provisional Application No. 61/784,901, filed March 14, 2013, which applications are orated herein by reference in their entirety.

BACKGROUND OF THE INVENTION Proliferative diseases, such as cancer, pose a serious challenge to society. Cancerous growths, including malignant cancerous growths, possess unique characteristics such as uncontrollable cell proliferation resulting in, for example, unregulated growth of ant tissue, an ability to invade local and even remote tissues, lack of differentiation, lack of able symptoms and most significantly, the lack of effective therapy and prevention.

Cancer can develop in any tissue of any organ at any age. The etiology of cancer is not y defined but mechanisms such as genetic susceptibility, chromosome ge disorders, viruses, environmental factors and immunologic disorders have all been linked to a malignant cell growth and transformation. Cancer asses a large category of medical conditions, affecting millions of individuals worldwide. Cancer cells can arise in almost any organ and/or tissue of the body. ide, more than 10 n people are diagnosed with cancer every year and it is estimated that this number will grow to 15 million new cases every year by 2020. Cancer causes six million deaths every year or 12% of the deaths worldwide.

SUMMARY OF THE INVENTION Provided herein are polynucleotide sequences encoding mutated forms of thymidine kinase from a human herpes simplex virus (HSV-TK), wherein the encoded HSV-TK is mutated at amino acid residue 25, 26, 32, 33, 167, 168 or a combination thereof, wherein the polynucleotide sequence is mutated ed to a polynucleotide sequence of SEQ ID NO: 1 or A polynucleotide sequence encoding a d form of thymidine kinase from a human herpes simplex virus (HSV-TK), n the encoded HSV-TK is mutated at amino acid residue , 26, 32, 33, 167, 168 or a combination thereof, wherein the polynucleotide sequence is mutated compared to a polynucleotide sequence of SEQ ID NO: 3. In one embodiment, the encoded HSV-TK is mutated at amino acid residues 167, 168, or a combination thereof to a polar, non-polar, basic or acidic amino acid. In another embodiment, the encoded HSV-TK is mutated at amino acid residue 167 to a polar, non-polar, basic or acidic amino acid. In yet another embodiment, the encoded HSV-TK is mutated at amino acid residue 168 to a polar, non- polar, basic or acidic amino acid. In still another embodiment, the encoded HSV-TK is mutated at both amino acid residues 167 and 168 to a polar, non-polar, basic or acidic amino acid.

In one embodiment, amino acid e 167 of the encoded HSV-TK is mutated to serine or phenylalanine. In another embodiment, amino acid residue 168 of the encoded HSV-TK is mutated to an amino acid selected from the group consisting of: histidine, lysine, cysteine, serine, and phenylalanine. In still another embodiment, the encoded HSV-TK is mutated at amino acids 25 and 26. In yet r embodiment, amino acid residues 25 and 26 are mutated to an amino acid chosen from the group ting of: glycine, serine, and glutamic acid. In another embodiment, the encoded HSV-TK is d at amino acid residues 32 and 33. In one ment, the amino acid residues 32 and 33 are mutated to an amino acid chosen from the group consisting of: glycine, serine, and glutamic acid. In one embodiment, the encoded HSV- TK is mutated at amino acid residues 25, 26, 32 and 33. In another embodiment, amino acid residues 25, 26, 32 and 33 are mutated to an amino acid chosen from the group consisting of: glycine, , and glutamic acid. In still another embodiment, the d HSV-TK comprises at least one mutation chosen ﬁom the group consisting of amino acid residues 25, 26, 32 and 33, and at least one mutation chosen from the group consisting of amino acid residues 167 and 168.

In still other embodiments, the encoded HSV-TK ce ﬁarther comprises a nuclear export signal (NES). In another embodiment, the nuclear export signal sequence is inserted at or ’ terminus of the HSV-TK near the 5 sequence. In another embodiment, the nuclear export signal ce is LQKKLEELELDG (SEQ ID NO: 24). In one embodiment, the encoded mutant HSV-TK does not localize exclusively to the nuclear region.

In one embodiment, the encoded modiﬁed HSV-TK exhibits a reduced amount of thymidine kinase activity as compared to ype HSV-TK. In another embodiment, the ty of the encoded modiﬁed HSV-TK is reduced by about 1.5 fold, about 2-fold, about 5- fold, about d, about 20-fold, about 30-fold, or about 50-fold. In still another embodiment, the activity of the encoded modiﬁed HSV-TK is reduced by about 1.5%, about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100%.

In one embodiment, the encoded HSV-TK comprises ons at amino acid residues , 26, 32, 33 and 168. In another embodiment, the encoded HSV-TK comprises mutations R25G, R26S, R32G, R33S and A168H.

In one embodiment, modiﬁed cleotide sequence comprises a nucleic acid sequence set forth as any one of SEQ ID NOS: 12-22. In still another embodiment, the modiﬁed WO 53258 polynucleotide sequence comprises a nucleic acid sequence set forth as any one of SEQ ID NOS: 16-22. In one embodiment, the sequence comprises TKl68dmNES (SEQ ID NO: 18). In still another embodiment, the polynucleotide encodes a modiﬁed HSV-TK polypeptide.

In still other embodiments, the polynucleotide further comprises a polynucleotide sequence coding for a second polypeptide, wherein said second polypeptide is a therapeutic polypeptide. In still other embodiments, the second therapeutic polypeptide is a second suicide gene or a growth . In some embodiments, the grth factor is chosen from the group consisting of epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), opoietin, G-CSF, GM-CSF, TGF-(x, TGF-B and fibroblast growth factor. In some embodiments, the second suicide gene is chosen from the group consisting of: a cytosine deaminase, a VSV-tk, IL-2, nitroreductase (NR), ylesterase, beta-glucuronidase, cytochrome p450, alactosidase, diphtheria toxin A-chain (DT-A), carboxypeptide G2 (CPGZ), purine nucleoside phosphorylase (PNP), and ytidine kinase (dCK).

In some ments, the polynucleotides ﬁarther comprises a polynucleotide encoding for a PiT-2 polypeptide. In still other embodiments, the polynucleotides disclosed herein further comprises a polynucleotide encoding for a targeting polypeptide. In one embodiment, the targeting polypeptide binds to an extracellular protein. In another embodiment, the extracellular protein is collagen.

Also provided herein are methods of killing neoplastic cells in a subject in need thereof, the method sing administering a therapeutically ive amount of a retroviral particle, the iral vector encoding an HSV-TK modiﬁed peptide as described herein.

In some embodiments, the retroviral particle is administered intravenously, intramuscularly, subcutaneoustly, intra-arterially, intra-hepatic arterially, intra-thecally, intraperitoneally and/or intra-tumorally. In other embodiments, the retroviral particle is administered intra-tumorally or enously. In yet other embodiments, the iral vector particle is stered intra-arterially.

In other embodiments, at least 1 x 1012 TVP of retroviral vector is administered cumulatively to the subject in need thereof In still other embodiments, at least 1 x 109 TVP of retroviral vector is administered at one time to the t in need thereof In still other embodiments, the prodrug is administered between about 1-2 days after administration of the retroviral vector particle. In some embodiments, the prodrug is chosen from the group consisting of ganciclovir, valganciclovir In , aciclovir, clovir, penciclovir. some ments, the prodrug is ganciclovir.

Also provided herein are methods for treating cancer in a patient in need f, the method comprising delivering a therapeutically effective amount of a retroviral vector particle, the retroviral vector encoding an HSV-TK modiﬁed peptide as described herein, followed by administration of a nucleoside prodrug to the patient in need thereof.

Also provided herein are methods of increasing HSV-TK ganciclovir, valganciclovir , aciclovir, clovir, penciclovir-mediated killing of neoplastic cells in a subject, the method comprising delivering a therapeutically effective amount of a iral vector particle comprising an HSV-TK to the subject in ction with a gap junction intracellular communication (GJIC)—increasing treatment. In some embodiments, the GJIC-increasing treatment comprises delivering a polynucleotide sequence encoding at least one gap junction subunit. In other embodiments, the gap on subunit is connexin 43, connexin 30, or connexin 26. In yet other embodiments, the gap junction subunit is a gap junction subunit modiﬁed to prevent anslational ations. In still other embodiments, the GJIC- increasing treatment comprises ring a polynucleotide sequence encoding E-cadherin. In still other embodiments, the GJIC-increasing treatment comprises delivering to the subject a compound from the group consisting of: gemcitabine; cAMP; a retinoic acid; a carotenoid; a glucocorticoid, a ﬂavanoid, apigenin, or lovastatin. In yet other embodiments, the GJIC- increasing treatment comprises proteasome inhibition. In one ment, the proteasome inhibition comprises administration ofN—Acetyl-Leu-Leu-Nle-CHO (ALLN) and/or chloroquine. In other embodiments, the ncreasing treatment comprises radiation or electrical ent. .

Also provided herein are methods of killing a cell, the method comprising: a) introducing into the cell a polynucleotide sequence according to any one of claims 1-26; b) ng or initiating the cell to express the expressed thymidine kinase or variant thereof; an c) ting the cell with an agent that is converted by thymidine kinase to a cytotoxic agent.

In one embodiment, a polynucleotide sequence encodes a mutated form of thymidine kinase from a human herpes simplex virus (HSV-TK) sing 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modiﬁcations. In another embodiment, a polynucleotide sequence encodes a mutated form of thymidine kinase from a human herpes simplex virus (HSV-TK) comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modiﬁcations. In another embodiment, a polynucleotide sequence s a mutated form of thymidine kinase from a human herpes simplex virus (HSV-TK) comprises 3, 4, 5, 6, 7, 8, 9, 10 or more modiﬁcations. In r ment, a polynucleotide sequence encodes a d form of thymidine kinase from a human herpes simplex virus (HSV-TK) comprises 4, 5, 6, 7, 8, 9, 10 or more ations. In another embodiment, a polynucleotide sequence encodes a mutated form of thymidine kinase from a human herpes simplex virus (HSV-TK) comprises 5, 6, 7, 8, 9, 10 or more modiﬁcations.

In one ment, the encoded HSV-TK may be mutated at amino acid residues 167, 168, or a combination thereof to a polar, non-polar, basic or acidic amino acid. For example, the encoded HSV-TK may be mutated at amino acid e 167 to a polar, non-polar, basic or acidic amino acid. In another example, the encoded HSV-TK may be mutated at amino acid residue 168 to a polar, non-polar, basic or acidic amino acid. In another example, the encoded HSV-TK may be mutated at both amino acid residues 167 and 168 to a polar, lar, basic or acidic amino acid.

In another ment, amino acid residue 167 of the d HSV-TK may be mutated to serine or phenylalanine.

In another embodiment, amino acid residue 168 of the encoded HSV-TK may be mutated to an amino acid selected from the group consisting of: histidine, lysine, cysteine, serine, and phenylalanine.

In another embodiment, the encoded HSV-TK may be mutated at amino acids 25 and 26.

For example, amino acid residues 25 and 26 may be d to an amino acid chosen from the group consisting of: glycine, serine, and glutamic acid.

In another embodiment, the encoded HSV-TK may be mutated at amino acid residues 32 and 33. For example, amino acid residues 32 and 33 may be mutated to an amino acid chosen from the group consisting of: glycine, serine, and glutamic acid.

In another embodiment, the encoded HSV-TK may be mutated at amino acid es , 26, 32 and 33. For example, amino acid residues 25, 26, 32 and 33 may be mutated to an amino acid chosen from the group consisting of: glycine, serine, and glutamic acid.

In another embodiment, the encoded mutant HSV-TK does not localize exclusively to the nuclear region.

In another embodiment, the encoded modiﬁed HSV-TK ts a reduced amount of ine kinase actiVity as compared to Wild-type .

In another embodiment, the thymidine kinase actiVity of the encoded modiﬁed HSV-TK may be d by about 1.5 fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about -fold, or about 50-fold.

In another embodiment,the thymidine kinase actiVity of the encoded modiﬁed HSV-TK may be reduced by about 1.5%, about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100%.

In another embodiment, the thymidine kinase actiVity of the encoded modiﬁed HSV-TK may be increased by about 1.5 fold, about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 30-fold, or about 50-fold.

In another embodiment, the thymidine kinase activity of the encoded modiﬁed HSV-TK may be increased by about 1.5%, about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100%.

Provided herein is polynucleotide sequence as described above, where the encoded HSV- TK comprises the mutation A167F, A168H or both.

A polynucleotide ce described herein may further comprise a polynucleotide sequence coding for a second polypeptide, where said second polypeptide is a therapeutic polypeptide. The therapeutic polypeptide may, in some instances, be a suicide gene. Suicide genes include, but are not limited to, a cytosine deaminase, a VSV-tk, IL-2, nitroreductase (NR), carboxylesterase, beta-glucuronidase, cytochrome p450, beta-galactosidase, diphtheria toxin A- chain (DT-A), carboxypeptide G2 , purine nucleoside phosphorylase (PNP), guanylate kinase, and ytidine kinase (dCK).

In one embodiment, a modiﬁed polynucleotide sequence bed herein may comprise a nucleic acid sequence set forth as any one of SEQ ID NOS: 12-24.

In another embodiment, a modiﬁed polynucleotide sequence described herein may comprise a nucleic acid sequence set forth as any one of SEQ ID NOS: 22-24.

In one embodiment, a polynucleotide sequence bed herein comprises a nuclear export signal. For example, a polynucleotide sequence may comprise HSV-TKA168HdmNES (SEQ ID NO: 18).

In another embodiment, a retroviral vector for use in the methods described herein comprises one or more splice site modiﬁcations.

In another embodiment, a retroviral vector for use in the methods described herein comprises HSV-TK A167Fsm, wherein ‘sm’ refers to the single mutation pair 26S (SEQ ID NO: 13) .

In another embodiment, a iral vector for use in the s described herein comprises HSV-TK m (SEQ ID NO: 12).

In another ment, a retroviral vector for use in the methods described herein comprises HSV-TK A167de, wherein ‘dm’ refers to the double mutation pair R25G-R26S, R32G-R33S (SEQ ID NO: 17).

In another embodiment, a retroviral vector for use in the methods described herein comprises HSV-TK m (SEQ ID NO: 16).

In another embodiment, a retroviral vector for use in the methods described herein comprises HSV-TK A167de and a nuclear export sequence d from mitogen-activated protein kinase kinase, an example of which is SEQ ID NO: 19.

In r embodiment, a retroviral vector for use in the methods described herein comprises HSV-TK Al68Hdm and an NES (SEQ ID NO: 18). In such an embodiment, the ce ses HSV-TK Al68H.

In another embodiment, a retroviral vector for use in the methods described herein comprises a HSV-TK, wherein such vector comprises an upgraded substrate binding domain and a mNLS/NES set. Examples of this exemplary embodiment include SEQ ID NOS: l8 and 19.

In another embodiment, a retroviral vector for use in the methods described herein comprises a HSV-TK, wherein the vector comprises a selectable marker, a glowing gene and/or one or more kill genes.

In another embodiment, a retroviral vector for use in the methods described herein comprises two modiﬁcations.

In another embodiment, a retroviral particle comprises a PiT-2 polynucleotide sequence and the retroviral particle speciﬁcally binds to a PiT-2 receptor on the surface of the target cells, thereby ng for uptake of the retroviral particle into the cell.

In another ment, a retroviral vector for use in the methods described herein comprises a HSV-TK, wherein the amino acid sequence encoded by the polynucleotide sequence comprises TKl68dmNES. ed herein is a method of increasing FHBG (9-[4-ﬂuoro (hydroxymethyl)butyl]guanine), FHPG (9u{[3~fluorou l uhydroxyulZmpropoxy]methyl)guanine), FGCV (ﬂuoroganciclovir), FPCV (ﬂuoropenciclovir), FIAU (l-(2'-deoxy-2'-ﬂuoro-l-B-D- arabinofuranosyl)iodouracil), FEAU (ﬂuoro-S-ethyl-l-beta-D-arabinoﬁ1ranosyluracil), FMAU (ﬂuoro-S-methylbeta-D-arabinofuranosyluracil), FHOMP -ﬂuoro hydroxypropanyloxy)methyl)-5 -methylpryrimidine-2,4( l H,3H)—dione), ganciclovir, valganciclovir, acyclovir, valacivlovir, penciclovir, radiolabeled dine with 4-hydroxy (hydroxymethyl)butyl side chain at N-l (HHG-S-FEP) or 5-(2-)hydroxyethyl)- and 5-(3- hydroxypropyl)-substituted pyrimidine tives bearing hydroxypropyl, acyclovir-, ganciclovir- and penciclovir-like side -mediated g of neoplastic cells in a subject, the method comprising delivering a eutically effective amount of vector particles encoding HSV-TK to the subject in conjunction with a gap junction ellular communication (GJIC)— increasing treatment.

In one embodiment, the HSV-TK used in such methods may be encoded by any of the polynucleotide sequences described herein.

The GJIC-increasing treatment may comprise, for example, delivering a polynucleotide sequence encoding at least one gap on subunit. A gap junction subunit may be, for example, connexin 43, connexin 30, or connexin 26. The gap on subunit may be a gap junction subunit modiﬁed to prevent posttranslational modiﬁcations.

In one embodiment, the GJIC-increasing treatment comprises delivering a polynucleotide sequence encoding E-cadherin.

In another embodiment, the GJIC-increasing treatment comprises delivering to the subject a compound from the group consisting of: gemcitabine; cAMP; a retinoic acid; a carotenoid; a glucocorticoid, a ﬂavanoid, apigenin, or lovastatin.

In another embodiment, the GJIC-increasing ent comprises some inhibition.

Proteasome inhibition may comprise administration ofN—Acetyl-Leu-Leu-Nle-CHO (ALLN) and/or chloroquine.

In another embodiment, the ncreasing treatment comprises radiation or photodynamic treatment, including coadministration with oxidative agents and agents that activate MAP kinases.

In another embodiment, the GJIC-increasing treatment comprises electrical treatment. ed herein is a method of killing a cell, the method sing: (a) introducing into the cell a polynucleotide sequence described herein; (b) allowing or ting the cell to express the expressed thymidine kinase or t thereof; and (c) contacting the cell with an agent that is converted by thymidine kinase to a cytotoxic agent.

Provided herein is a method of increasing thymidine kinase bystander effect, the method comprising delivering a sequence ng a gap junction subunit in ction with a iral vector particle encoding HSV-TK. In some embodiments, the retroviral les may be targeted to a cell or system of interest. In some embodiments, the retroviral targeting method may comprise the incorporation of a factor that recognizes or binds to the cell or system of st. In some embodiments, the retroviral targeting method may comprise the incorporation of targeting proteins, including binding to proteins or receptors on the e of the cell of system of interest, including antibodies, receptor binding proteins or proteins that bind to cellular components, including but not limited to collagen. In some embodiments the targeting protein may comprise proteins that bind to collagen, ing but not limited to peptides, proteins and/or protein domains that include a collagen binding domain.

BRIEF DESCRIPTION OF THE DRAWINGS The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and ages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are ed, and the accompanying drawings of which: Figure l exempliﬁes how HSV-TK splice site l avoids an inactivated form of HSV-TK. PCR analysis of T-cell lines and primary T cells transduced with HSV-TK s with (HuT SF/Tk/mut) or without (HuT GlTkl SvNa) splice site removal.

Figure 2 provides an exemplary schematic for a Phase IA clinical trial with a composition described herein.

Figure 3 provides an provides an ary schematic for a Phase IB clinical trial with a composition described herein.

Figure 4 provides an provides an exemplary schedule of events for Phase IA clinical trial for cohorts l to 3.

Figure 5 provides an provides an exemplary schedule of events for Phase IA clinical trial for cohorts 4 and above.

Figure 6 provides an es an exemplary schematic for a Phase IB al trial with a composition described .

Figure 7 provides an provides an exemplary schematic for a Schedule A of clinical trial for treatment with a composition described herein.

Figure 8 provides an provides an exemplary schematic for a Schedule B of clinical trial for treatment with a composition bed herein.

Figure 9 provides an illustration of a PiT-2 transmembrane molecule. The box represents the approximate location of an Anti-PiT-2 Western antibody binding site.

Figure 10 provides an illustration of a PiT-2 transmembrane molecule. The box represents the approximate location of an Anti-PiT-2 IHC antibody binding site.

Figure 11 provides exemplary Reximmune constructs with s HSV-TK modiﬁcations. Figure llA: GM-CSF Minus, HSV-TK l67sm. Figure llB: GM-CSF Minus, HSV-TK l68sm. Figure 110 GM-CSF Minus, HSV-TK l67dm. Figure llD: GM-CSF Minus, HSV-TK l68dm. Figure llE: GM-CSF Minus, HSV-TK l67dm + NES. Figure llF: GM-CSF Minus, HSV-TK l68dm + NES.

Figure 12: K, Protein Detection By Western is for the retroviral vectors shown in Figure 11. Viral DNA was ected into 293T Vector Producer Cells, the cells were lysed, HSV-TK proteins were detected with an anti-HSV-TK antibody. All of the HSV-TK viral vectors were found to express high levels of HSV-TK protein.

Figure 13 provides exemplary retroviral vectors. Figure 13A: RexRed-TK A168H.

Figure 13B: RexRed-TK l67-dm. Figure 13C: RexRed-TK 168 dm.

Figure 14 provides onal exemplary retroviral vectors where a ular form of codon optimation was employed.

Figure 14A: RexRed-TK l67-dm + NES. Figure 14B: RexRed-TK l68-dm + NES.

Figure 14C: -TK l67-dm + NES JCO. Figure 14D: RexRed-TK l68-dm + NES JCO.

JCO = justiﬁed codon optimization.

Figure 15 provides additional exemplary retroviral vectors. Figure 15A: -TK Al68F. Figure 15B: RexRed-TK Al68F (GCV speciﬁc). Figure 15C: Rex-Hygro-R-TK Al68F containing the hygromycine resistance gene.

Figure 16 provides additional exemplary retroviral s. Figure 16A: Rex-Hygro-R- TK Al67F. Figure 16B: Q-PiT-2 is a vector containing a viral receptor gene that binds to a PiT- 2 receptor on the surface of target cells.

Figure 17 provides additional exemplary retroviral vectors. Figure 17A: Original une-C. Figure 17B. Reximmune-C containing an e with a mTK39 (HSV- TKSR39) kill gene with neomycin resistance gene (NeoR) and selectable marker inserted.

Figure 18 provides an exemplary of Reximmune-C + a mutated bacterial cytidine deaminase (mBCD) kill gene.

Figure 19 provides an ary of Reximmune-C + a mutated yeast ne deaminase (mYCD) kill gene.

Figure 20 illustrates one example of a RexRed Super TK which includes a g gene (RFP) and a kill gene that contains the identiﬁed sequences at the noted positions.

Figure 21 provides an illustration of retroviral s having an updated substrate binding domain and +/- mNLS and/or +/-NES set, highlighting the sequence differences between Reximmune-Cl or 2, SR-39 and the Wildtype HSV-TK gene, and having installed a second therapeutic gene in place of the RFP gene between the LTR and SV40 promoters Figure 22 illustrates RexRed Super TK Al67F which includes a glowing gene (RFP) and a kill gene that contains the noted sequences at positions 159-161 and 167-169.

Figure 23 provides exemplary retroviral vectors that are Reximmune-C multicolor clones of LNCE A375 transduced cells. Figure 23A: LNC-EGFP which contains an enhanced green ﬂuorescent protein as a glowing gene. Figure 23B: RexRed which contains a red ﬂuorescent protein as a glowing gene.

Figure 24 es exemplary vectors that a glowing gene only or a hygromycin resistance gene selectable marker only.

Figure 25: Tk-GCV kill results in parent and PiTCHO-Kl lines. The graphs illustrate the data for a single RxC2-transduction protocol. The same batch of RxC2 was used for all experiments (titer imately 5E+lO total virus particles per milliliter (TVP) as determined by reverse transcriptase in tandem with quantitative polymerase chain reaction (RT-qPCR)).

Figure 25A: GCV kill of RxC2-transduced CHO-Kl parent line after 4 days in GCV (4 doses).

Figure 25B: GCV kill of RxC2-transduced PiTCHO-Kl after 4 days in GCV (4 doses).

Figure 26: Tk-GCV kill in parent and PiTCHO-Kl following a Triple RxC2- transduction ol. Figure 26A: GCV kill of RxC2-triple transduced CHO-Kl parent on day 9 (10% plate, 5 doses GCV). Figure 26B: GCV kill of riple transduced PiTCHO-Kl on day 9 (10% plate, 5 doses GCV).

Figure 27: illustrates TK-GCV kill after triple transduction with Reximmune-C2 (HSV- HdmNES) (SEQ ID NO: 18) in a MIA-PaCa-2 human pancreatic carconima cell line.

GCV kill of RxC2-triple transduced MIA-PaCa2, 25% of initial cells reseeded, day 8, with various concentrations of GCV.

Figure 28 Illustrates TK-GCV kill after triple transduction of PiTMIA-PaCa-2 cells with Reximmune-C2. GCV kill of RxC2-triple transduced PiTMIA-PaCa2, 25% of initial cells, day 8with various concentrations of GCV.

Figure 29: Graphic results from a bystander in vitro assay where human melanoma A375 Hygro TK clones were treated with 20 mM GCV.

Figure 30: Graphic results from a der in vitro assay where C6-Hygro-TK clones were treated with 20 mM GCV.

Figure 31 is a graph depicting the percentage of GCV kill after une-C2 triple transduction of various cancer cell lines..

Figure 32 rates a graph of RxC2-tranduced CHO-Kl cell lines after four days in GCV.

Figure 33 illustrates a graph of RxC2-tranduced HA-CHO-Kl cell lines after four days in GCV.

Figure 34 illustrates immuno histochemistry (IHC) ofHSV-TK sub cell Localization in 293T cells Transient Transfection, 24 hour Primary AB (Santa Cruz) with RexCl HSV-TK (left panel) and RexC2 HSV-TK (right panel) DETAILED DESCRIPTION OF THE INVENTION HSV-TK gene therapeutic products are available, but are non-optimal with t to maximal gene expression and tumor kill activity both in vitro and in viva including cancer gene therapy.

Disclosed herein for the first time is an optimization of codons within HSV-TK genes to produce improved suicide genes with enhanced pro-drug activation mance in the context 2014/029814 of a viral or psuedoviral gene delivery system. The optimized gene delivery system insures both l HSV-TK pro-drug enzyme activity and production of high titers of viral particles.

Thus, disclosed herein is the optimization of candidate optimized HSV-TK genes prepared using both bioinformatics software and custom analysis by the present inventors utilizing knowledge of the functions and limitations of the genes and viral vector system.

The ing zation steps represent exemplary methods that were utilized by the t inventors to arrive at the embodiments described herein. Software ed codon zation may be utilized to remove rare and low use codons to improve HSV-TK protein expression. The GC content within the newly codon optimized gene may be adjusted to avoid gene synthesis and other ms.

Known splice or and splice donor sequences within HSV-TK may be removed.

Tracts of poly-pyrimidines, particularly those introduced by codon optimization which may be involved in splicing may be removed.

One single strong Kozak translation initiation sequence may be included in front of the start codon (ATG) while possible Kozak sequences within HSV-TK open reading frame may be removed. Some of these sequences may have been introduced by codon optimization and it would be understood that modifications may need to be made in multiple ions to optimize a gene for improved tumoricidal activity.

Nuclear Localization Sequences (NLSs) within HSV-TK may be removed to export expressed HSV-TK wherein the expressed HSV-TK protein is not localized exclusively to the nucleus, but instead accumulates in the cytoplasm.

Restriction sites ﬂanking HSV-TK gene making it possible to clone the gene into many locations in the disclosed retroviral vectors may be added, while excluding these same restriction sites within the HSV-TK gene itself.

A double stop codon at end of HSV-TK gene may be included to insure complete termination of HSV-TK translation.

Mutations near the substrate g domain at amino acid locations 159-161 within the HSV-TK gene may be evaluated.

Mutants in the substrate binding domain at amino acid location 167 within the HSV-TK gene may be evaluated for increased enzyme activity s the ug nucleoside analogue, such as gangciclovir and similar pro-drugs, as well as selectivity for their ability to kill cancer cells.

Mutants in the substrate binding domain at amino acid location 168 within the HSV-TK gene may be evaluated for increased ug GCV enzyme activity and selectivity for their ability to kill cancer cells.

] Mutants in the substrate binding domain at amino acid location 167 + 168 within the HSV-TK gene may be evaluated for increased pro-drug GCV enzyme activity and selectivity for their ability to kill cancer cells.

The use of tags, fusion proteins and linkers of HSV-TK to other genes and proteins may be evaluated.

Further methods of optimization may also be considered for use in the methods described herein. Once a gene is optimized in this way, its gene sequence can be sent to a gene synthesis company for custom gene synthesis.

DEFINITIONS Unless deﬁned otherwise, all technical and scientiﬁc terms used herein have the same meaning as is ly understood by one of skill in the art to which the invention(s) belong. All patents, patent applications, published ations and publications, GenBank sequences, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. In the event that there are a plurality of deﬁnitions for terms herein, those in this section prevail. Where reference is made to a URL or other such identiﬁer or address, it understood that such identiﬁers can change and particular information on the intemet can come and go, but equivalent ation can be found by searching the intemet. nce thereto evidences the availability and public dissemination of such information.

As used , “nucleic acid” refers to a polynucleotide containing at least two covalently linked nucleotide or nucleotide analog subunits. A c acid is generally a ibonucleic acid (DNA), a ribonucleic acid (RNA), or an analog of DNA or RNA. Nucleotide s are commercially available and methods of preparing polynucleotides containing such nucleotide analogs are known (Lin et al. (1994) Nucl.

Acids Res. 22:5220-5234; Jellinek et al. (1995) Biochemistry 34: 1 1363-1 1372; is et al. (1997) Nature Biotechnol. 15 :68-73). The nucleic acid is generally single-stranded, double- stranded, or a mixture thereof. For purposes herein, unless speciﬁed ise, the c acid is double-stranded, or it is apparent from the context.

As used herein, “DNA” is meant to include all types and sizes ofDNA molecules including cDNA, plasmids and DNA ing modiﬁed nucleotides and nucleotide analogs. 2014/029814 As used herein, “nucleotides” include nucleoside mono-, di-, and triphosphates.

Nucleotides also include modiﬁed nucleotides, such as, but are not limited to, phosphorothioate nucleotides and deazapurine nucleotides and other nucleotide analogs.

The term “polynucleotide” as used herein means a polymeric form of nucleotide of any length, and includes ribonucleotides and deoxyribonucleotides. Such term also includes single-and double-stranded DNA, as well as single-and double-stranded RNA. The term also includes modiﬁed polynucleotides such as methylated or capped cleotides.

As used herein, the term “subject” refers to animals, , insects, and birds into which the large DNA molecules are introduced. Included are higher organisms, such as s and birds, including humans, primates, rodents, cattle, pigs, rabbits, goats, sheep, mice, rats, guinea pigs, cats, dogs, horses, chicken and others.

As used herein, “administering to a subject” is a procedure by which one or more delivery agents and/or large nucleic acid molecules, together or tely, are introduced into or applied onto a subject such that target cells which are present in the subject are eventually contacted with the agent and/or the large c acid molecules. [001 19] As used herein, ery vector” or “delivery vehicle” or “therapeutic ” or peutic system” refers to both viral and non-viral particles that harbor and transport exogenous nucleic acid molecules to a target cell or tissue. Viral vehicles include, but are not limited to, retroviruses, adenoviruses, iral viruses, herpes s and adeno-associated viruses. Non-viral vehicles include, but are not limited to, articles, nanoparticles, virosomes and liposomes. “Targeted,” as used herein, refers to the use of ligands that are associated with the delivery vehicle and target the vehicle to a cell or tissue. Ligands include, but are not limited to, antibodies, receptors and collagen-binding domains.

As used herein, “delivery,” which is used interchangeably with “transduction,” refers to the process by which exogenous nucleic acid les are transferred into a cell such that they are d inside the cell. Delivery of nucleic acids is a distinct process from expression of nucleic acids.

As used herein, a “multiple g site (MCS)” is a c acid region in a plasmid that contains multiple restriction enzyme sites, any of which can be used in ction with standard recombinant technology to digest the vector. “Restriction enzyme digestion” refers to catalytic cleavage of a nucleic acid molecule with an enzyme that ﬁanctions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are cially available. Use of such enzymes is widely understood by those of skill in the art.

Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector. 2014/029814 As used herein, “origin of replication” (often termed “ori”), is a c nucleic acid ce at which ation is initiated. Alternatively an autonomously replicating sequence (ARS) can be employed if the host cell is yeast.

As used , “selectable or screenable markers” confer an identiﬁable change to a cell permitting easy identiﬁcation of cells containing an expression vector. Generally, a selectable marker is one that confers a ty that allows for selection. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drug ance marker.

Usually the ion of a drug selection marker aids in the cloning and ﬁcation of transformants, for example, genes that confer ance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated. In some ments, screenable enzymes such as herpes x virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) are utilized. One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the c acid encoding a gene product. Further examples of selectable and screenable markers are well known to one of skill in the art.

The term “transfection” is used to refer to the uptake of foreign DNA by a cell.

A cell has been “transfected” when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al., Virology 52:456 (1973); Sambrook et al., Molecular Cloning: A tory Manual (1989); Davis et al., Basic Methods in Molecular Biology ; Chu et al., Gene 13: 197 (1981). Such techniques can be used to introduce one or more exogenous DNA moieties, such as a nucleotide integration vector and other nucleic acid molecules, into suitable host cells. The term captures chemical, electrical, and viral-mediated transfection procedures.

As used herein, “expression” refers to the process by which nucleic acid is translated into peptides or is transcribed into RNA, which, for e, can be translated into peptides, polypeptides or proteins. If the nucleic acid is derived from genomic DNA, sion includes, if an appropriate eukaryotic host cell or organism is selected, splicing of the mRNA.

For heterologous nucleic acid to be expressed in a host cell, it must initially be delivered into the cell and then, once in the cell, ultimately reside in the nucleus.

As used herein, a “therapeutic course” refers to the periodic or timed administration of the vectors disclosed herein within a deﬁned period of time. Such a period of time is at least one day, at least two days, at least three days, at least ﬁve days, at least one week, at least two weeks, at least three weeks, at least one month, at least two months, or at least six months. Administration could also take place in a chronic manner, z'.e., for an undeﬁned period of time. The periodic or timed administration includes once a day, twice a day, three times a day or other set timed stration.

As used , the terms “co-administration, 3, (Cadministered in combination with” and their grammatical equivalents or the like are meant to encompass administration of the selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are administered by the same or different route of administration or at the same or different times. In some embodiments, a therapeutic agent as sed in the t application will be co-administered with other agents. These terms encompass administration of two or more agents to an animal so that both agents and/or their metabolites are present in the animal at the same time. They include simultaneous administration in separate compositions, administration at different times in separate compositions, and/or administration in a composition in which both agents are present. Thus, in some ments, a therapeutic agent and the other agent(s) are stered in a single composition. In some ments, a therapeutic agent and the other agent(s) are admixed in the composition. In further embodiments, a therapeutic agent and the other agent(s) are administered at separate times in separate doses.

The term “host cell” s, for example, microorganisms, yeast cells, insect cells, and ian cells, that can be, or have been, used as recipients for multiple constructs for ing a delivery vector. The term includes the progeny of the original cell which has been transfected. Thus, a “host cell” as used herein generally refers to a cell which has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to l, accidental, or deliberate mutation.

As used herein, “genetic therapy” involves the transfer of heterologous DNA to the certain cells, target cells, of a mammal, particularly a human, with a disorder or conditions for which therapy or diagnosis is sought. The DNA is introduced into the selected target cells in a manner such that the heterologous DNA is expressed and a therapeutic product encoded thereby is produced. In some ments, the heterologous DNA, directly or indirectly, mediates expression ofDNA that s the therapeutic product. In some embodiments, the heterologous DNA s a product, such as a peptide or RNA that mediates, directly or -l6- 2014/029814 indirectly, expression of a therapeutic product. In some embodiments, genetic therapy is used to deliver a c acid encoding a gene product to e a defective gene or supplement a gene product produced by the mammal or the cell in which it is introduced. In some embodiments, the introduced c acid encodes a therapeutic compound, such as a growth factor or inhibitor thereof, or a tumor necrosis factor or inhibitor thereof, such as a receptor therefore, that is not lly produced in the mammalian host or that is not produced in therapeutically effective amounts or at a therapeutically useful time. In some embodiments, the heterologous DNA encoding the therapeutic product is modiﬁed prior to introduction into the cells of the afﬂicted host in order to enhance or otherwise alter the product or expression thereof.

As used herein, ologous nucleic acid sequence” is generally DNA that encodes RNA and proteins that are not normally produced in vivo by the cell in which it is expressed or that mediates or encodes mediators that alter expression of nous DNA by affecting ription, translation, or other regulatable biochemical processes. Any DNA that one of skill in the art would recognize or consider as heterologous or foreign to the cell in which it is expressed is herein encompassed by heterologous DNA. Examples of heterologous DNA include, but are not limited to, DNA that encodes traceable marker proteins, such as a protein that confers drug resistance, DNA that encodes therapeutically effective substances, such as anti-cancer agents, enzymes and es, and DNA that s other types of proteins, such as antibodies. In some ments, antibodies that are encoded by heterologous DNA is secreted or expressed on the surface of the cell in which the heterologous DNA has been uced.

As used herein, the term “thymidine kinase ” refers to not only the c protein described herein (as well as the nucleic acid sequences which encode these proteins), but derivatives thereof which may include various structural forms of the primary protein which retain biological activity.

As used herein, “unmutated thymidine kinase” refers to a native or wild-type thymidine kinase polypeptide sequence.

As used herein, “suicide gene” refers to a nucleic acid encoding a product, wherein the product causes cell death by itself or in the present of other compounds.

As used herein, the term “mutated” or “replaced by another nucleotide” means a nucleotide at a certain position is replaced at that on by a nucleotide other than that which occurs in the unmutated or previously mutated sequence. That is, in some instances, speciﬁc ations may be made in different nucleotides. In some embodiments, the replacements are made such that the relevant splice donor and/or acceptor sites are no longer present in a gene. See, e.g., Figure l.

As used herein, a “polar amino acid” refers to amino acid residues Asp(N), Cys (C), Gln (Q), Gly (G), Ser (S), Thr (T) or Tyr (Y).

As used herein, a “non-polar amino acid” refers to amino acid residues Ala (A), Ile (1), Leu (L), Met (M), Phe (F), Pro (P), Trp (W), or Val (V).

As used herein, a “basic amino acid” refers to amino acid residues Arg (R), His (H), or Lys (K).

] As used herein, an “acidic amino acid” refers to amino acid residues Asp (D) or Glu (E).

IMPROVED HSV-TK Thymidine kinase is a salvage pathway enzyme which phosphorylates natural nucleoside substrates as well as nucleoside analogues. Generally, viral thymidine kinase is exploited therapeutically by administration of a nucleoside analogue such as ganciclovir or acyclovir to a cell expressing Viral thymidine kinase, wherein the Viral thymidine kinase phosphorylates the nucleoside analogue, creating a toxic product capable of killing the cell.

Polynucleotide sequences encoding viral thymidine kinase of the present invention may be ed from a wide variety of Viral ine kinases. In some ments, the Viral thymidine kinase mutant is derived from Herpesvirz'dae ine kinase including, for example, both primate herpes viruses, and non-primate herpes viruses such as avian herpes viruses. Representative examples of suitable herpes viruses include, for e, Herpes Simplex Virus (HSV) Type 1, Herpes Simplex Virus Type 2, Varicella zoster Virus, marmoset herpes virus, feline herpes virus type 1, pseudorabies virus, equine herpes virus type 1, bovine herpes virus type 1, turkey herpes virus, Marek's disease virus, herpes virus saimir and Epstein-Barr Virus.

Herpes s may be y obtained from commercial sources such as the American Type Culture tion ”, Rockville, Md.). viruses may also be isolated from naturally ing courses (e.g., an infected animal).

IMPROVEMENTS TO TK GENE Disclosed herein, in some embodiments, is a polynucleotide sequence encoding HSV-TK. In some ments, the polynucleotide sequence encodes a wild-type HSV-TK amino acid sequence. In some embodiments, the polynucleotide sequence encodes a mutated HSV-TK amino acid sequence.

Exemplary ures that may be used in preparation of an optimized polynucleotide sequence provided herein include, but are not limited to: codon optimization; correction of splice sites, removal of poly-pyrimidine tracts and excess GC content; addition of -l8- WO 53258 single Kozak sequence, removal of unwanted Kozak sequences; inclusion of restriction sites for ning into retroviral or other vectors; removal of nuclear localization sequences or addition of nuclear export sequences; addition of mutation sequences; addition of double stop codon sequences; addition of tags, linkers and fusion sequences; preparation of sequence ﬁle for submission to gene sis company; subcloning of synthesized gene into retroviral vectors; ion of cent protein genes into retroviral vectors; inclusion of selectable marker genes into retroviral vectors; preparation of Maxiprep d DNA; transfection of retroviral producer or other cells; lab, pilot or GMP scale production of retrovirus; transduction of target cells with retrovirus; GCV or analogus ug mediated cell kill assay; Hypoxanthine/Aminopterin/ Thymidine (HAT) selection assay; able marker drug ion procedure to e iral transduced cell lines; ﬂuorescent microscopy and photography to detect and document retroviral transduced target cells; quantitative ﬂuorescent detection of retroviral transduced target cells; Western protein expression assay; other procedures and assays as needed for HSV-TK analysis; or a combination thereof. Protocols for such methods are described herein, are commercially available or are described in the public literature and ses.

In some embodiments, described herein is a method of obtaining an improved HSV-TK ce. In some embodiments, the method comprises: a) correction and/or removal of splice sites; and/or b) adjustment to a single Kozak sequence. Optionally, in some embodiments, the method further comprises inclusion of restriction sites for sub-cloning of the HSV-TK sequence. Optionally, or in addition, in some embodiments, the method further comprises removal of nuclear localization sequences.

Provided herein is a cleotide sequence ng a mutated form of thymidine kinase from human simplex virus (HSV-TK), n the encoded HSV-TK is mutated at amino acid residue 25, 26, 32, 33, 167, 168, or a combination thereof, wherein the polynucleotide sequence is mutated compared to a polynucleotide sequence of SEQ ID NO: 1 or 3. In such sequences, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, l2, 13, or 14 mutations may be made.

Provided herein is a polynucleotide sequence encoding a mutated form of thymidine kinase from human simplex virus (HSV-TK), wherein the encoded HSV-TK is mutated at amino acid residue 25, 26, 32, 33, 167, 168, or a combination thereof, wherein the polynucleotide sequence is mutated compared to a polynucleotide sequence of SEQ ID NO: 1.

In such sequences, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, l2, 13, or 14 mutations may be made.

Provided herein is a polynucleotide sequence encoding a mutated form of thymidine kinase from human simplex virus (HSV-TK), wherein the encoded HSV-TK is mutated at amino acid residue 25, 26, 32, 33, 167, 168, or a ation thereof, wherein the polynucleotide sequence is mutated compared to a polynucleotide sequence of SEQ ID NO: 3.

In such sequences, 1,2, 3,4, 5, 6, 7, 8, 9, 10, ll, 12, 13, or 14 mutations may be made.

Modiﬁcations may be conservative or non-conservative mutations. A mutation may be made such that the d amino acid is modiﬁed to a polar, non-polar, basic or acidic amino acid.

Provided herein is a polynucleotide sequence encoding a d form of viral thymidine kinase from human simplex virus (HSV-TK), wherein the encoded HSV-TK includes a nuclear export sequence. Provided herein is a cleotide sequence encoding a mutated form of thymidine kinase from human simplex virus (HSV-TK), where the encoded HSV-TK is improved in function compared to wild-type HSV-TK and comprises A168H dmNES (Q system-QMV er properly fused to LTR promoter regions), where NES refers to a nuclear export sequence. In one embodiment, a mutant HSV-TKA168HdmNES is a mutant HSV-TK gene for inclusion in Reximmune-C2. In one embodiment, the NES is derived from MAP Kinase Kinase (MAPKK). In yet another embodiment, the cleotide sequence for NES is CTGCAGAAAAAGCTGGAAGAGCTGGAACTGGATGGC (SEQ ID NO: 23). In other embodiments, the NES polypeptide sequence is LQKKLEELELDG (SEQ ID NO: 24).

In some embodiments, disclosed herein are mutations to a polynucleotide sequence encoding Human Simplex Virus Thymidine Kinase (HSV-TK) wherein mutations are not made to the polypeptide ce of wildtype . tide positions are referred to by reference to a position in SEQ ID NO: 1 (wildtype (wt) HSVl-TK nucleotide sequence) or SEQ ID NO: 3 (HSV-TK in Reximmune-C HSV-TK; SR39 mutant and R25G-R26S Mutation of the HSV-TK nuclear localization signal (NLS)).

In one ment, a Sac I-Kpn I restriction sites bounding the le double stranded oligonucleotides of the mutant HSV-TK SR39 mutant region is provided. See, for example, SEQ ID NOS: 6 and 7, where the Sac I and Kpn I sites are shown on the left and right, respectively. Bold, underlining illustrates the sites where mutations may be made. SEQ ID NOS: 8 and 9 illustrate an exemplary sequence after cutting with Sac I and Kpn I. Exemplary forward and reverse primers that may be used to make the mutations are shown as SEQ ID NOS: and 11.

Exemplary zed HSV-TK polynucleotide sequences are provided, for example, as SEQ ID NOS: l2-24.

However, when such references are made, the ion is not intended to be limited to the exact sequence as set out in SEQ ID NO: 1 or 3, but includes ts and derivatives thereof. Thus, identification of nucleotide locations in other thymidine kinase sequences are contemplated (i.e., identiﬁcation of nucleotides at positions which the d person would consider to correspond to positions recited in SEQ ID NO: 1 or 3).

In some embodiments, tides are replaced by taking note ofthe c code such that a codon is d to a different codon which codes for the same amino acid residue.

In some embodiments, nucleotides are replaced within coding s of a HSV-TK encoding nucleic acid ce, yet the nucleic acid sequence maintains wild type HSV-TK protein expression.

In some embodiments, codons are mutated to such that the encoded HSV-TK ts increased activity. In some embodiments, the codon GCT is used to represent alanine.

In some embodiments, the codon AGA is used to represent arginine. In some embodiments, the codon AAT is used to represent asparagine. In some embodiments, the codon GAT is used to represent aspartic acid. In some embodiments, the codon TGT is used to represent cysteine. In some embodiments, the codon CAG is used to represent glutamine. In some embodiments, the codon GAA is used to represent glutamic acid. In some embodiments, the codon GGA is used to represent e. In some embodiments, the codon CAT is used to represent histidine. In some ments, the codon ATT is used to represent isoleucine. In some embodiments, the codon CTG is used to represent e. In some embodiments, the codon AAA is used to represent lysine. In some embodiments, the codon ATG is used to represent methionine. In some embodiments, the codon TTT is used to represent phenylalanine. In some embodiments, the codon CCT is used to represent proline. In some embodiments, the codon TCT is used to represent serine. In some embodiments, the codon ACA is used to represent threonine. In some embodiments, the codon TGG Is used to represent tryptophan. In some embodiments, the codon TAT is used to represent tyrosine. In some embodiments, the codon GTG is used to represent valine. In some embodiments, the codon TGA is used as a stop codon. Exemplary codon positions for mutation are ed in the following table.

Improved Codon Usage for Designing Human Genes, First Choice Codon Optimization which Reduces G/C content n Aspartic Acid (Asp) ( D ) GAT Cysteine ( Cys) ( C ) TGT [- Glutaminewmw 7 Glutamic Acid ( Glu) ( E ) GAA n Glycine ( Gly) ( G) GGA n ine (His) (H) CAT Isoleucine ( Ile) (I) ATT 11 Leucine ( Leu) ( L ) 12 Lysine ( Lys) ( K) 13 Methonine (Met) (M ) 14 Phenylalanine ( Phe ) ( F) TTT Proline ( Pro ) ( P ) CCT 16 Serine (Ser) ( S ) TCT 17 Threonine ( Thr) ( T ) ACA 18 Tryptophan ( Trp) ( W) 19 ne ( Tyr) (Y ) TAT Valine (Val ) (V) 21 Stop (Term) ( * ) TGA In such embodiments, 5/21 codons contain “C or G” in third position (24%); 0/21 codons contain “C” in third position (0 %); 5/21 codons contain “G” in third position (24%); and 16/21 codons n “A or T” in third on (76%).

In yet other embodiments, about 3-7 codons of 21 codons contain “C or G” in the third position; above 0-3 codons of 21 codons contain “C” in the third position; about 3-7 codons of 21 codons contain “G” in the third position; and about 14-18 codons of 21 codons contain “A or T” in the third on.

In some embodiments, the codon GCA is used to represent alanine. In some embodiments, the codon AGG is used to represent arginine. In some embodiments, the codon AAC is used to represent asparagine. In some embodiments, the codon GAC is used to represent aspartic acid. In some embodiments, the codon TGC is used to represent cysteine. In some embodiments, the codon CAA is used to represent glutamine. In some ments, the codon GAG is used to represent ic acid. In some embodiments, the codon GGC is used to represent glycine. In some embodiments, the codon CAC is used to represent histidine. In some embodiments, the codon ATC is used to represent isoleucine. In some embodiments, the codon CTC is used to represent leucine. In some embodiments, the codon AAG is used to represent . In some embodiments, the codon ATG is used to represent methionine. In some embodiments, the codon TTC is used to represent phenylalanine. In some embodiments, the codon CCA is used to represent proline. In some embodiments, the codon AGC is used to represent serine. In some embodiments, the codon ACT is used to represent threonine. In some embodiments, the codon TGG is used to represent tryptophan. In some embodiments, the codon TAC is used to represent tyrosine. In some embodiments, the codon GTC is used to ent valine. In some embodiments, TAA is used as a stop codon.

Improved Codon Usage for Designing Human Genes, 2nd Choice Codon Optimization which Reduces G/C content Am 11 cm 12 M9 14 W 16 A62 19 ”2 GT2 21 W In such embodiments, 16/21 codons contain “C or G” in third position (76%); 11/21 codons contain “C” in third on (52 %); 5/21 codons contain “G” in third position (24%); and 5/21 codons contain “A or T” in third on (24%).

In yet other embodiments, about 14-18 codons of 21 codons contain “C or G” in the third position; about 9-13 codons of 21 codons contain “C” in the third position; about 3-7 codons of 21 codons contain “G” in the third position; and about 3-7 codons of 21 codons contain “A or T” in the third position.

In some embodiments, the following rare codons are are avoided if possible, unless changing the rare codon sequence creates new splice acceptor and/or alternate Kozak sites or adds an unwanted restriction site or other problematic seqeunce, within the coding region of a polynucleotide encoding mutant HSV-TK, or a variant thereof: GCG for alanine; CGA or CGT for arginine; TTA or CTA for e; CCG for e; TCG for serine; ACG for threonine; and GTA for valine. Rare codons to be avoided if possible are those that have a codon/a.a./fraction per codon per a.a. less than or equal to 0.12.

Rare Codon ‘44 USU C 0.45 ‘50 US“ C 0.54 .30 US$.* 0.47 "1: _.. .24 USS W 1.00 000 L 013 0.00 P 0.23 an H 0 V 0 000 L 0.20 0.0.: P 0.32 0ch H 0.33 000 R 0.13 CCP P 0.23 0P0 a 0,2? 000 L 0,40 0P0 a 0J3 000 R 0.20 P00 I 0.30 P00 T 0.23 PPU 110.4": P00 3 0.15 P00 :1: 0.47 P00 T 0.30 3"}: £10.53 PP: 3 0.24 PUP. I 0.1"! P0 .T 0.23 PPP K 0.03 P0P R 021 P00 M 1.00 m PPS K 0.37 .000 R 021 000V0.10 000A I: F0.. --.1 mL‘lr-IEl 0.46 SSH E 0.15 000 v 0.24 000 P 0.40 mmw5293’- HHUU CI. m “:3. EEC E 0.34 EBA P I2!._ MI La 0.42 GEE-‘1 5 0.225 000' 0' 0.40 Em 0.53 GEE G 0.225 E [Eﬁdcnfa.amffraction per sedan pEr 0.0,] “ HBED a data frﬁm the Sauna Usage ﬁatabaae In some embodiments, altering codons as described herein results in about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or greater tage increase in activity.

High tage Codon Optimization was found to improve protein expression but increases GC gene t. Codon optimization was assessed and determined to have the following characteristics.

Hﬁamen: ﬁrsﬁes‘hy LimaWm ‘ V hyﬂbmth Fs'e. suem“ s . base. WsiE-asémss Awaﬁi‘s mm g’ A3533 i B ‘} Agar?mm * and i" B ‘ \ H 2: I anim at: Luci; {L} {uterine :ef' Lire} {' L 3 {mains :'? Lire} i L} ] Because of the unsatisfactory results obtained with fully ted codon optimization software programs, customized codon optimization was performed to increase both n expression and titers obtained. The initial step includes the use of a codon optimizer program as a ﬁrst screen in order to set each codon for the correct reading frame to that most preferred in the subject species, including humans, giving a ‘raw’ codon optimization.

Generally, any desired cloning restriction sites are excluded from use during this stage of the 13100688.

The results are further reﬁned by editing DNA ces in a DNA editor program, and searching for degenerate codons, such as pyrimidines (e.g., by searching for “Y” codons). The following operations are then performed, in this order: Manual search of the sequence for runs of “Y,” generally at least ﬁve or more “Y” sequential runs. These sequences are highlighted in a given sequence, and the DNA editor program is used to determine a translation that includes the DNA sequence listed in register to the peptide sequence to insure that changes to codons do not affect the translated protein.

Each codon in a run of 5 or more Y’s is evaluated. When available, the wobble base of each codon is converted to the most favorable A-G base for the amino acid (usually an adenine), and the result ed. If the result of the change creates a purine-rich run ending at ’ AG, the changes or near a 3 are manually reversed. If there is no most favorable A-T base available for the wobble base or it causes another sequence conﬂict, the the most favorable C-G base is used for the wobble base.

If the result is a rare codon (< 10% usage), that codon is moved to the next available codon in the frame.

If another codon change can ablate the putative acceptor site, changes are made to revert to the al sequence. If no such alternative change is available, then the original alteration is implemented.

Once this process is complete, the sequence is examined 5’ to 3’ for alternate reading frames. At each reading frame, the 5 bases 3’ of the ATG codon are examined for their suitability as Kozak sequences. If the ATG gives a methionine in the reading frame of the desired gene, s are limited to ablating the Kozak sequence, ﬁrst by converting the wobble base of the “- ” wobble base to the ATG to a “T” (if possible), then the “-4” wobble base.

In rare cases, it may be desirable to convert the second codon in the g frame, if ally an “AGN” base (Ser/Arg) to a codon ing in T (for ) or C (for arginine). The situation is generally not encountered when strictly ng the above algorithm however, as the “AGN” codons are avoided due to the “AG” sequence pair.

In cases where the alternate reading frame differs from that of the message, AND the Kozak ce surrounding it ﬁts the consensus “CCACCatgG”, the wobble base of the in- frame codon is altered to remove the start codon. This generally happens (but not always) as a result of the codon optimization and/or splice acceptor ablation process.

In-process checks are lly performed to ensure that the peptide sequence is ged. At the ﬁnal check stage, if there are too many ‘rare’ codons in use (generally 2 or more) it may be desirable to prioritize which are used, with preference to changes given to the longer pyrimidine runs from the ‘raw’ codon optimized ce. Finally, any needed restriction sites are added, and a last check is performed to insure that the polypeptide is unchanged from the original sequence before the optimization process is begun and that any desired restriction sites remain unique to those that are added for cloning purposes.

Splice Site Modiﬁcation Introns are generally spliced out ofRNA in order to join exons. A splice donor site is a site in RNA on the 5' side of the RNA which is removed during the splicing process and which contains the site which is cut and rejoined to a nucleotide residue within a splice acceptor site. Thus, a splice donor site is the on between the end of an exon and the start of the intron. Generally, a splice donor site in RNA is the eotide GU (or a GT dinucleotide in the corresponding DNA sequence).

A splice acceptor site is a site in RNA on the 3' side of the RNA which is removed during the splicing process and which contains the site which is cut and ed to a tide residue within a splice donor site. Thus, a splice acceptor site is the junction between the end of an intron (typically terminating with the dinucleotide AG) and the start of the downstream exon.

In some embodiments, disclosed herein is a nucleic acid sequence encoding a thymidine kinase n at least one nucleotide corresponding to a splice donor site is replaced by r nucleotide. See, e.g., Figure l (Chalmers et al., Mol. Ther. 4:146-8 (2001)). In further embodiments, the tides of the splice acceptor sites are not altered. In some embodiments, at least one nucleotide ponding to a splice acceptor site is replaced by another nucleotide.

In some embodiments, disclosed herein is a nucleic acid sequence encoding a thymidine kinase wherein at least one of the nucleotides corresponding to splice donor site nucleotides at positions 329 and 330 of a polynucleotide sequence (e.g., SEQ ID NO: 1 or 3) is ed by another nucleotide. In some embodiments, both of the nucleotides at positions 327 and 555 are ed by other nucleotides. For example, position 327 may be mutated to an amino acid residue selected from: G to A. Altemately, or in addition, position 555 may be mutated to an amino acid residue selected from: G to A. In one embodiment, the modiﬁed HSV-TK has a polynucleotide ce of SEQ ID NO: 18 in which HSV-TK was improved in the following ways: HSV-TK NESdmNLS Al68H, CO & SC NES = nuclear export sequence from MAP Kinase Kinase (MAPKK) dmNLS = double mutated HSV-TK Nuclear Localization Sequence CO = codon zed SC = splice donor/acceptor site corrected at 327 and 555, Underlined sequence 2014/029814 SEQ ID NO: 18 gtCaGCGGCCGCACCGGTACGCGTCCACCATGGCCCTGCAGAAAAAGCTGGAAGAGCTGGAACTGGATGG CCCCGGCCACCAGCACGCCAGCGCCTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGC GCaCTGCGgCCaGGATCTCAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCC TGCTGCGCGTGTACATCGACGGaCCaCACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCT GGGCAGCCGCGACGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAG ACCATCGCCAACATCTACACCACCCAGCACCGCCTGGACCAEGGCGAGATCAGCGCCGGCGACGCCGCCG TGGTGATGACCAGCGCCCAGATtACaATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGCaCCaCA CATCGGCGGCGAGGCCGGCAGCAGCCACGCaCCaCCaCCaGCaCTGACCCTGATCTTCGACCGgCACCCa ATCGCaCACCTGCTGTGCTACCCgGCaGCaCGCTACCTGATGGGCtCCATGACaCCaCAEGCCGTGCTGG CCTTCGTGGCCCTGATCCCaCCaACaCTGCCCGGCACCAACATCGTGCTGGGCGCCCTGCCCGAGGACCG CCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAGCGCCTGGACCTGGCCATGCTGGCCGCCATC CGCCGCGTGTACGGCCTGCTGGCCAACACCGTGCGCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACT GGGGCCAGCTGAGCGGCACCGCCGTGCCaCCaCAGGGCGCCGAGCCaCAGAGCAACGCCGGaCCaCGaCC aCACATCGGCGACACCCTGTTCACCCTGTTCCGgGCaCCaGAGCTGCTGGCaCCaAACGGCGACCTGTAC AACGTGTTCGCCTGGGCCCTGGACGTGCTGGCCAAGCGCCTGCGCtCCATGCACGTGTTCATCCTGGACT ACGACCAGtcaCCgGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGT GACaACaCCCGGCAGCATCCCaACaATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGGCC AACTAATAGGGATCCCTCGAGAAGCTTgtca In some embodiments, disclosed herein is a nucleic acid sequence encoding a thymidine kinase wherein at least one of the nucleotides corresponding to splice acceptor site nucleotides at ons 554 and 555, or at least one of the nucleotides ponding to splice acceptor site nucleotides at positions 662 and 663, or at least one of the nucleotides corresponding to splice acceptor sites at positions 541 and 542 of the Wild type sequence is replaced by another nucleotide. For example, position 541 may be mutated to an amino acid residue selected from: G to A. Position 542 may be mutated to an amino acid residue selected from: G to A. Position 554 may be mutated to an amino acid residue selected from: G to A.

Position 555 may be d to an amino acid residue selected from: G to A. Position 662 may be d to an amino acid residue selected from: G to A. Position 663 may be mutated to an amino acid e selected from: G to A.

In some embodiments, at least one of the nucleotides of the wild-type HSV-TK encoding sequence is replaced as described in Table 1 below.

TABLE 1 Position Mutation 84 843 c —> A 846 C a A 879 C a G 882 C a A 168 885 c HA 171 897 c —>A 378 —m1_ C HA 961 AHT Position Mutation Position Mutation 420 C—>A 962 G—>C A Kozak sequence ﬂanks the AUG start codon Within mRNA and inﬂuences the recognition of the start codon by eukaryotic ribosomes. In some embodiments, a polynucleotide sequence ng HSV-TK comprises no more than one Kozak sequence. In some embodiments, the Kozak ce is am of the coding portion of the DNA sequence. In some embodiments, the Kozak sequence of a polynucleotide encoding HSV-TK is modiﬁed to produce a Kozak sequence with a higher efﬁciency of translation initiation in a mammalian cell.

In some ments, modiﬁcation of the Kozak sequence does not produce an amino acid substitution in the encoded HSV-TK polypeptide product. In some embodiments, modiﬁcation of the Kozak sequence results in at least one amino acid substitution in the d HSV-TK polypeptide product. In one embodiment, the modiﬁed HSV-TK has a polynucleotide sequence of SEQ ID NO: 18.

In some embodiments, a polynucleotide sequence encoding HSV-TK comprises a modiﬁcation that inserts one or more restriction sites. The optimal site for insertion of one or more restriction sites may be ined empirically and/or using a computer program to analyze the sequence. In one non-limiting embodiment, a ﬁrst restriction site is inserted ' end of upstream of the Kozak and ATG start site and a second restriction site is inserted at the 3 the sequence. See, for example, SEQ ID NO: 18, underlined section below.

HSVTK NESdmNLS Al68H, CO & SC NES = nuclear export sequence from MAPKK dmNLS = double mutated Nuclear Localization Sequence CO = codon optimized SC = splice corrected at 327 and 555, previously bed Kozak Sequence, usly described Restriction Sites, ined and specified as: CGC ACCGGT ACGCGT = Not-I, Age-I, and MLU-I) (GGATCC CTCGAG AAGCTT = SamH-", Xho-I and Pind- ) SEX)IDIVO:18 gtcaGCGGCCGCACCGGTACGCGTCCACCATGGCCCTGCAGAAAAAGCTGGAAGAGCTGGAACTGGATGG CAGCTACCCCGGCCACCAGCACGCCAGCGCCTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGC AGCACCGCaCTGCGgCCaGGATCTCAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCC TGCTGCGCGTGTACATCGACGGaCCaCACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCT GGGCAGCCGCGACGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAG GCCAACATCTACACCACCCAGCACCGCCTGGACCAaGGCGAGATCAGCGCCGGCGACGCCGCCG TGGTGATGACCAGCGCCCAGATtACaATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGCaCCaCA CATCGGCGGCGAGGCCGGCAGCAGCCACGCaCCaCCaCCaGCaCTGACCCTGATCTTCGACCGgCACCCa ATCGCaCACCTGCTGTGCTACCCgGCaGCaCGCTACCTGATGGGCtCCATGACaCCaCAaGCCGTGCTGG CCTTCGTGGCCCTGATCCCaCCaACaCTGCCCGGCACCAACATCGTGCTGGGCGCCCTGCCCGAGGACCG CCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAGCGCCTGGACCTGGCCATGCTGGCCGCCATC CGCCGCGTGTACGGCCTGCTGGCCAACACCGTGCGCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACT GGGGCCAGCTGAGCGGCACCGCCGTGCCaCCaCAGGGCGCCGAGCCaCAGAGCAACGCCGGaCCaCGaCC aCACATCGGCGACACCCTGTTCACCCTGTTCCGgGCaCCaGAGCTGCTGGCaCCaAACGGCGACCTGTAC AACGTGTTCGCCTGGGCCCTGGACGTGCTGGCCAAGCGCCTGCGCtCCATGCACGTGTTCATCCTGGACT ACGACCAGtcaCCgGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGT GACaACaCCCGGCAGCATCCCaACaATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGGCC AACTAATAGGGATCCCTCGAGAAGCTTgtca Other splice site modiﬁcations are disclosed in the examples below and are considered for inclusion as a modiﬁed TK sequence that can be used in the claimed methods.

In some embodiments, the polynucleotide sequence encoding HSV-TK comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 60, 75, 80, 85, 90, 95, 100 or more codon substitutions. In some embodiments, the cleotide sequence encoding HSV-TK comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 60, 75, 80, 85, 90, 95, 100 or more codon tutions, wherein the codon substitutions comprise the substitution of a codon having a higher frequency of usage in a ian cell than the wild type codon at that position.

However, in some embodiments, less favored codons may be chosen for individual amino acids depending upon the particular situation.

In some embodiments, the polynucleotide sequence encoding HSV-TK sing at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 60, 75, 80, 85, 90, 95, 100 or more codon substitutions has less than about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 or 3 wherein the sequence identity is determined over the ﬁll length of the coding sequence using a global alignment . In some embodiments, the corresponding encoded polypeptide sequence has at least 75 %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a HSV-TK amino acid sequence, e.g., SEQ ID NO: 2 or 4.

In some embodiments, the cleotide sequence ng HSV-TK comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 60, 75, 80, 85, 90, 95, 100 or more codon substitutions, wherein the codon substitutions comprise the substitution of a codon having the highest frequency of usage in a mammalian cell for the wild type codon at that position. In some ments, the corresponding encoded ptide sequence has at least 75 %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a HSV-TK amino acid sequence, e.g., SEQ ID NO: 2 or 4.

In some embodiments, the polynucleotide ce encoding HSV-TK comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 60, 75, 80, 85, 90, 95, 100 or more codon substitutions, wherein the substituted codons have a frequency of usage greater than or equal to about 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35 or higher. In some embodiments, the corresponding encoded polypeptide sequence has at least 75 %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a HSV-TK amino acid sequence, e.g., SEQ ID NO: 2 or 4.

In some embodiments, the polynucleotide sequence encoding HSV-TK comprises less than about 45, 40, 35, 30, 25, 20 or fewer codons, wherein the codons have a frequency of usage less than about 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24 or 0.25. In some embodiments, the corresponding d polypeptide sequence has at least 75 %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a HSV- TK amino acid sequence, e.g., SEQ ID NO: 2 or 4.

In some embodiments, the polynucleotide sequence encoding HSV-TK ses at least 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or more of codons having a frequency of usage greater than or equal to about 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, or higher. In some embodiments, the corresponding encoded ptide sequence has at least 75 %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a HSV-TK amino acid sequence, e.g., SEQ ID NO: 2 or 4.

In some embodiments, the polynucleotide ce encoding HSV-TK ses at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or more of codons haVing the t frequency of usage in a mammalian cell. In some embodiments, the corresponding encoded polypeptide sequence has at least 75 %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a HSV-TK amino acid sequence, e.g., SEQ ID NO: 2 or 4.

In some embodiments, the polynucleotide sequence encoding HSV-TK ses less than about2l%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10% or less of codons haVing a frequency e less than about 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24 or 0.25. In some embodiments, the polynucleotide sequence comprises less than about2l%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10% or less of codons haVing a frequency ofusage less than about 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24 or 0.25 in a mammalian cell. In some embodiments, the corresponding encoded polypeptide sequence has at least 75 %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a HSV-TK amino acid sequence, e.g., SEQ ID NO: 2 or 4.

In some embodiments, the polynucleotide sequence encoding HSV-TK comprises codon substitutions, wherein at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the codons have been changed as compared to the wild type sequence. In some embodiments, the polynucleotide sequence encoding HSV-TK comprises codon substitutions, wherein at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more ofthe codons have been changed to a codon having a higher frequency of usage in a mammalian cell as compared to the wild type sequence. In some embodiments, the ponding encoded ptide sequence has at least 75 %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a HSV-TK amino acid ce, e.g., SEQ ID NO: 2 or 4.

In some embodiments, the polynucleotide sequence encoding HSV-TK comprises codon substitutions, wherein at least 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the codons have been changed to a codon having the highest frequency of usage in a mammalian cell as ed to the wild type sequence. In some embodiments, the corresponding encoded ptide ce has at least 75 %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% ce identity to a HSV-TK amino acid sequence, e.g., SEQ ID NO: 2 or 4.

Non-Conserved Mutations The Viral thymidine kinase gene from the selected herpesvirus may be readily isolated and mutated as described below, in order to construct nucleic acid molecules encoding a thymidine kinase enzyme comprising one or more ons which increases biological actiVity of the thymidine kinase, as compared to unmutated wild-type thymidine kinase. The biological actiVity of a thymidine kinase may be readily determined utilizing any of the assays known in the art, including for example, determination of the rate of nucleoside analogue uptake or determination of the rate of nucleoside or nucleoside analogue phosphorylation. In addition, thymidine kinase mutants may be readily selected which are terized by other biological properties, such as thermostability and protein stability.

In some embodiments, the polynucleotide sequence encoding HSV-TK is modiﬁed to remove or modify a predicted signal sequence. In some embodiments, the polynucleotide is d to remove or modify a nuclear localization sequence (NLS). In some embodiments, the polynucleotide is d to remove the nuclear localization sequence. In some embodiments, the cleotide is modified to modify the NLS so that if no longer functions to localize HSV-TK exclusively to the nucleus.

In some embodiments, a HSV-TK polypeptide sequence is d at amino acid residues 167, 168, or both. In one example, the sequence is mutated at amino acid residue 167.

In another example, the sequence is mutated at amino acid residue 168. In another example, the WO 53258 sequence is mutated at amino acid residues 167 and 168. Amino acid e 167 may be mutated to serine or phenylalanine. Amino acid residue 168 may be mutated to histidine, lysine, cysteine, serine or alanine. In some embodiments, a HSV-TK polypeptide sequence is mutated at amino acid residues 25 and/or 26. In amino acid residues 25 and/or 26 may be mutated to an amino acid chosen from the group consisting of: glycine, serine, and glutamic acid. In some embodiments, the HSV-TK polypeptide sequence is mutated at amino acid residues 32 and/or 33. Amino acid residues 32 and/or 33 may be mutated to an amino acid chosen from the group ting of: e, serine, and glutamic acid. In some embodiments, the HSV-TK polypeptide is mutated at amino acid residues 25, 26, 32, and/or 33. Amino acid residues 25, 26, 32, and/or 33, may be mutated to an amino acid chosen from the group consisting of: glycine, serine, and glutamic acid. Amino acid residue modiﬁcations may be made in comparison to a polypeptide sequence of SEQ ID NOS: 2 or 4.

In accordance with the present invention, mutant thymidine kinase enzymes which are encoded by the above-described nucleic acid molecules are provided, as well as vectors which are capable of expressing such molecules. In some embodiments, expression vectors are provided comprising a promoter operably linked to a nucleic acid molecule of the present invention. In some embodiments, the vector is a viral vector capable of directing the expression of a nucleic acid molecule. Representative examples of such viral vectors include herpes simplex viral vectors, adenoviral vectors, adenovirus-associated viral vectors, pox vectors, parvoviral vectors, baculovirus vectors and iral vectors. In some embodiments, viral vectors are provided which are capable of directing the expression of a c acid le which s a ine kinase enzyme comprising one or more mutations, at least one of the mutations encoding an amino acid substitution which increases a biological ty of thymidine kinase, as compared to ted (z'.e. , wild-type) thymidine kinase.

In some embodiments, a nucleic acid molecule provided herein encodes a thymidine kinase enzyme e of phosphorylating a nucleoside analogue at a level at least % greater than the level of phosphorylation of the nucleoside analogue by a wild-type thymidine kinase enzyme. In some embodiments, the thymidine kinase enzyme is capable of phosphorylating a nucleoside analogue at a level at least 15%, at least 20%, at least 25%, at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 500% greater than the level of phosphorylation of the nucleoside analogue by a wild-type thymidine kinase enzyme. Representative examples of suitable nucleoside analogues include gancyclovir, acyclovir, famciclovir, buciclovir, penciclovir, valciclovir, triﬂuorothymidine, eoxy, 2- ﬂuoro, beta-D-arabino furanosyl]iodouracil, ara-A, araT 1-beta-D-arabinofuranoxyl thymine, -ethyl-2'-deoxyuridine, -5'-amino-2, eoxyuridine, idoxuridine, AZT, AIU, 2014/029814 dideoxycytidine and AraC. In some embodiments, the improved TK mutant lacks thymidine kinase activity.

In some embodiments, the Km value for thymidine kinase activity of a disclosed HSV-TK mutant is at least 2.5 um. In some embodiments, the KIn value for thymidine kinase activity of a disclosed HSV-TK mutant is at least 5 um, at least 10 um, at least 15 um, at least um, at least 25 um, at least 30 um, at least 40 um, at least 50 um, at least 60 um, at least 70 um, at least 80 um, at least 90 um, at least 100 um, at least 150 um, at least 200 um, at least 250 um, at least 300 um, at least 400 um, at least 500 um, at least 600 um, at least 700 um, at least 800 um, at least 900 um, or at least 1000 um. In some embodiments, the t KIn of a disclosed HSV-TK mutant compared to wild-type HSV-TK is at least 15%, at least 20%, at least %, at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 500%.

Within one ment of the present invention, truncated derivatives of HSV- TK mutants are provided. For example, site-directed mutagenesis may be readily performed in order to delete the N—terminal 45 amino acids of a thymidine kinase mutant, thereby ucting a truncated form of the mutant which retains its ical activity.

Mutations in nucleotide sequences constructed for expression of derivatives of thymidine kinase mutants should preserve the g frame phase of the coding sequences.

Furthermore, the mutations will preferably not create complementary regions that could hybridize to produce secondary mRNA ures, such as loops or hairpins, which would adversely affect translation of the receptor mRNA. Such tives may be readily constructed using a wide variety of techniques, including those discussed above.

MODIFIED THYMIDINE KINASE MUTANTS Using the methods described herein, the inventors determined that the majority of the candidates for optimized HSV-TK genes appeared to be compatible with a retroviral expression system and produce biologically useful retroviral titers. rmore, the optimized HSV-TK genes which orated most of these optimizations (SEQ ID NO: 18 ted pro-drug GCV enzyme activity and selectivity for their ability to kill cancer cells following retroviral transduction ry. The mutant HSV-TK gene A168H, which was codon optimized and splice corrected appeared to have the highest GCV mediated cancer kill activity (SEQ ID NOs: 12, 16, 18, or 22). The same version of this HSV- TK gene A168H and mutated at amino acids 159-161 from LIF to IFL exhibited GCV mediated cancer cell kill activity.

The mutant HSV-TK gene Al67F (SEQ ID NOS: l3 ,17, or 19) which was codon optimized and splice corrected had very high GCV mediated cancer kill activity following retroviral transduction delivery, but more surprisingly had NO thymidine kinase activity as determined by expressing this gene ing retroviral transduction delivery in 3T3 TK(-) cells selected with HAT medium. To our knowledge, this is the most GCV selective HSV-TK synthetic gene product for GCV tion which has no Thymidine activity ( HAT assay) ever evaluated biologically.

The double mutant HSV-TK gene Al67F + Al68H (SEQ ID NO: 14) unexpectedly ablates both GCV and ine enzyme ty by exhibiting very little GCV mediated cancer kill activity and very little thymidine activity ( HAT assay), The present inventors identiﬁed that it is possible to produce functional HSV-TK fusions of genes such as bacterial ne deaminase, yeast cytosine deaminase, neomycin phosphotransferase and include linker ces and retain HSV-TK GCV mediated cancer cell killing activity.

In one embodiment, a codon optimized HSV-TK gene with GCV-mediated cancer killing activity may be made which retains one or more nuclear localization sequences which is not fused to one or more other therapeutic genes.

Additional modifications to and/or evaluations of an optimized HSV-TK gene described herein may include one or more of the following: removal ofknown nuclear localization sequences within HSV-TK; increased pro-drug GCV enzyme ty and selectivity for their ability to kill cancer cells, te the use of more tags, fusion proteins and linkers of HSV-TK to other genes and proteins, co-expression of HSV-TK optimized genes with other optimized suicide and cancer killer genes in cancer cells, include optimized HSV-TK genes in a Reximmune-C type retroviral vector ; tion and testing of a Reximmune-C type GMP product, or any combination thereof.

EXEMPLARY POLYNUCLEOTIDE SE UENCES In one embodiment, a polynucleotide ce described herein comprises a nuclear export signal. For example, a polynucleotide ce may comprise TKl68dmNES.

In another embodiment, a retroviral vector for use in the s described herein comprises one or more splice site modifications.

In another embodiment, a retroviral vector for use in the s described herein comprises HSV-TK Al67Fsm (SEQ ID NO: 13).

In another ment, a retroviral vector for use in the methods described herein comprises HSV-TK Al68Hsm (SEQ ID NO: 12).

In another ment, a iral vector for use in the methods described herein comprises HSV-TK Al67de (SEQ ID NO: 17).

In another embodiment, a retroviral vector for use in the methods described herein comprises HSV-TK Al68dm (SEQ ID NO: 16).

In another embodiment, a retroviral vector for use in the methods described herein comprises HSV-TK Al67de and an NES (SEQ ID NO: 19).

In another embodiment, a retroviral vector for use in the methods described herein comprises HSV-TK Al68Hdm and an NES (SEQ ID NO: 18). In such an embodiment, the sequence comprises HSV-TK A168H.

In another embodiment, a retroviral vector for use in the methods described herein comprises a HSV-TK, wherein such vector comprises an upgraded substrate g domain and a mNLS/NES set.

In another embodiment, a retroviral vector for use in the methods described herein comprises a HSV-TK, wherein the vector comprises a selectable marker, a glowing, ﬂuorescent or bioluminescent gene and/or one or more kill genes.

In another embodiment, a retroviral vector for use in the methods described herein comprises at least two ations.

CONSTRUCTION OF THYMIDINE KINASE MUTANTS ine kinase mutants of the present invention may be constructed using a wide variety of techniques. For example, mutations may be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, ﬂanked by ction sites ng ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence s a derivative having the d amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-speciﬁc (or t speciﬁc) mutagenesis procedures may be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required. Deletion or truncation derivatives of ine kinase mutants may also be constructed by ing convenient restriction endonuclease sites adjacent to the d deletion. Subsequent to restriction, overhangs may be ﬁlled in, and the DNA religated. Exemplary methods of making the alterations set forth above are disclosed by Sambrook et al. (Molecular g: A Laboratory Manual, 2Ild Ed., Cold Spring Harbor Laboratory Press, 1989).

Other derivatives of the thymidine kinase mutants disclosed herein include conjugates of thymidine kinase s along with other ns or polypeptides. This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion proteins which may be added to facilitate ation or identiﬁcation of thymidine kinase mutants (see US.

Pat. No. 4,851,341, see also, Hopp etaZ.,Bi0/Techn010gy 6:1204, 1988.).

IMPROVEMENT OF DIATED KILLING In some embodiments, the polynucleotide sequence encoding HSV-TK further comprises a sequence encoding a secondary therapeutic agent or polypeptide. In some embodiments, secondary therapeutic agent or ptide is a diagnostic or eutic agent or polypeptide.

In some embodiments, the secondary eutic agent or polypeptide is an additional “suicide protein” that causes cell death by itself or in the presence of other compounds. In some embodiments, the second suicide gene is chosen from the group including: penicillin-V-amidase, penicillin-G-amidase, beta-lactamase, carboxypeptidase A, linamarase (also referred to as B-glucosidase), the E. coli gpt gene, and the E. coli Deo gene, a cytosine deaminase, a VSV-tk, IL-2, nitroreductase (NR), ylesterase, beta-glucuronidase, cytochrome p450, beta-galactosidase, diphtheria toxin A-chain (DT-A), carboxypeptide G2 (CPG2), purine nucleoside phosphorylase (PNP), and deoxycytidine kinase (dCK).

In some embodiments, the second suicide protein converts a prodrug into a toxic compound. As used herein, “prodrug” means any compound useful in the methods disclosed herein that can be converted to a toxic t, z'.e., toxic to tumor cells. The prodrug is converted to a toxic product by the suicide n. Representative examples of such prodrugs include: FHBG (9-[4-ﬂuoro(hydroxymethyl)butyl]guanine), FHPG (9m{[3~fluoro»1“hydroxvu 2~propoxy]methyl)guanine), FGCV (ﬂuoroganciclovir), FPCV (ﬂuoropenciclovir), FIAU (1-(2'- deoxy-2'-ﬂuoroB-D-arabinoﬁ1ranosyl)iodouracil), FEAU (ﬂuoro-S-ethyl-l-beta-D- arabinofuranosyluracil), FMAU (ﬂuoro-S-methylbeta-D-arabinofuranosyluracil), FHOMP (6- ((1 -ﬂuorohydroxypropanyloxy)methyl)methylpryrimidine-2,4(1H,3H)—dione), ganciclovir, valganciclovir, acyclovir, valacivlovir, penciclovir, radiolabeled dine with 4- hydroxy(hydroxymethyl)butyl side chain at N-l (HHG-S -FEP) or 5-(2-)hydroxyethyl)- and 5- (3 -hydroxypropyl)-substituted pyrimidine derivatives bearing 2,3-dihydroxypropyl, acyclovir-, ganciclovir- and penciclovir-like side chains for thymidine ; ifosfamide for oxidoreductase; oxypurine arabinoside for VZV-TK; 5-ﬂuorocytosine for cytosine deaminase; doxorubicin for beta-glucuronidase; CB1954 and nitroﬁlrazone for nitroreductase; and N—(Cyanoacetyl)-L-phenylalanine or hloropropionyl)-L-phenylalanine for carboxypeptidase A.

] In some embodiments, the secondary therapeutic agent or polypeptide is chosen from the group including, but are not limited to, cell cycle control agents, agents which inhibit 2014/029814 cyclin proteins, such as antisense cleotides to the cyclin A and/ or D genes, growth factors such as, for example, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), erythropoietin, G-CSF, , TGF-u, TGF-B, and ﬁbroblast grth factor, cytokines, including, but not limited to, Interleukins 1 through 13 and tumor necrosis factors, anticoagulants, anti-platelet agents, anti-inﬂammatory agents, anti-angiogenic factors, tumor suppressor proteins, clotting factors, including Factor VII, Factor VIII and Factor IX, protein S, protein C, antithrombin III, von rand Factor, cystic fibrosis embrane tance regulator (CFTR), and negative selective markers.

] In some embodiments, a secondary eutic agent or polypeptide is a cancer suppressor, for example p53 or Rb, or a nucleic acid encoding such a protein or polypeptide.

Other examples of secondary therapeutic agents or polypeptides include pro- apoptotic therapeutic proteins and ptides, for example, p15, p16, or le/WAF-l.

In some embodiments, a secondary therapeutic agent or polypeptide is a cytokine.

Examples of nes include: GM-CSF locyte macrophage colony stimulating factor); TNF-alpha (Tumor is factor ; Interferons including, but not limited to, IFN-alpha and IFN—gamma; and eukins including, but not limited to, Interleukin-l (ILl), Interleukin- Beta (IL-beta), Interleukin-2 (IL2), Interleukin-4 (IL4), Interleukin-5 (IL5), Interleukin-6 (IL6), Interleukin-8 (IL8), Interleukin-10 (ILlO), Interleukin-l2 (IL12), Interleukin-l3 (ILl3), Interleukin- 1 4 (IL 1 4), Interleukin-15 (IL 1 5), Interleukin- 1 6 (IL 1 6), Interleukin-l 8 (ILl 8), Interleukin-23 , Interleukin-24 (IL24), although other embodiments are known in the art.

In some embodiments, the secondary therapeutic agent or polypeptide is pro- apoptotic. Examples of pro-apoptotic proteins or polypeptides include, but are not limited to: Bax, Bad, Bik, Bak, Bim, cytochrome C, apoptosis-inducing factor (AIF), Puma, CT 10- regulated kinase (CRK), Bok, glyceraldehydephosphate dehydrogenase, Prostate sis se Protein-4 (Par-4), Smac, Kinase C8, Fas, inhibitory PAS domain protein (IPAS), and Hrk.

In some embodiments, the secondary therapeutic agent or polypeptide is ed in cell to cell communication. In some embodiments, the secondary therapeutic agent or polypeptide is involved in gap cell junctions. In some embodiments, the secondary therapeutic agent or polypeptide is a connexin. In some embodiments, the therapeutic protein or polypeptide is a connexin chosen from the group connexin 43, connexin 32 and connexin 26.

In some embodiments, the secondary therapeutic agent or polypeptide is encoded by the human receptor gene PiT-2 (SLC20A2) . The ropic Envelope gene product included in the Reximmune-Cl and 2 retroviral vector binds to the PiT-2 receptor prior to target cell infection.In some embodiments, the secondary therapeutic agent or polypeptide is encoded by the human receptor gene PiT-l (SLC20A1) . The Gibbon Ape Luekemia Virus ( GALV) Envelope gene t binds to the PiT-l receptor prior to target cell infection.

In some embodiments, the secondary therapeutic agent or polypeptide is an N- terminal tion of a iral protein, wherein the N-terminal truncation comprises a functional or binding domain of the envelope protein.

INCREASING INTRACELLULAR COMMUNICATION TO IMPROVE TREATMENT Increase in Bystander Effect Disclosed herein, in some embodiments, is a method of sing the HSV-TK prodrug substrate bystander effect. As used herein, the “bystander effect” refers to the phenomenon by which a HSV-TK positive exerts a kill effect on neighboring HSV-TK negative cells following induction of expression of HSV-TK expression in the HSV-TK positive cells.

In some embodiments, is a method of sing the HSV-TK prodrug-mediated bystander effect, for e after treatment with GCV, in conjunction with increasing gap junction intracellular communication. In some embodiments, HSV-TK prodrug-mediate bystander effect increases the kill rate by 10%, by 20%, by 30%, by 40%, by 50%, by 60%, by 70%, by 80%, by 90% or by 100% or more.

Gap junctions are regions of the cell membrane with clusters of gap junction channels that directly connect the asm of one cell with the cytoplasm of another cell. A gap junction channel is composed of two hemichannels (connexons) ed by each of two neighboring cells. A connexon is comprised most often of six connexin proteins, which are a large family of proteins having a basic structure sing four transmembrane domains, two extracellular loops, and a cytoplasmic loops.

Gap junctions serve in various physiological roles, such as grth control and homeostasis (i.e., rapid equilibration of ions, nutrients, and ﬂuids between cells). In addition, gap junctions serve as ical synapses in cells that are able to propagate electrical signals, such as cardiac myocytes, smooth muscle cells, and neurons.

Once phosphorylated, GCV can travel through G] into adjoining cells that share the junctions. GCV-P will be phosphorylated ﬁarther in those cells and trigger cell death as in the HSK-TK expressing cell. The extend of the Bystander effect s on the existence of GAP ons and therefore it will differ between cell types. But see, Dahle et al. “Gap onal intercellular communication is not a major mediator in the bystander effect in photodynamic treatment of MDCKII cells.” ion Res. 154: 331-341 (Sept. 2000).

The viral TK enzyme is sensitive to the prodrug ganciclovir (GCV) which resembles the DNA base guanine.

When GCV is added to cell medium, the viral TK (but not the host non-viral TK) phosphorylates the GCV, converting it into a drug as, now phosphorylated, it will compete with dGTP for incorporation into DNA because of its similarity with guanine.

Incorporation will cause termination of the DNA chain synthesis. Transfer of GCV-monoP into non-cancer cells will not be toxic to them unless they are actively dividing.

The normal cells at risk are only those in close contact to the viral TK-expressing cells when treated with high levels of GCV drug.

Disclosed herein, in some embodiments, is a method of sing the viral thymidine-kinase mediated killing of target cells in a t, the method comprising ring vector les encoding HSV-TK in conjunction with gap junction intracellular communication (GJIC) —increasing ent. In some embodiments, the target cells are neoplastic cells. In some embodiments, the GJIC-increasing treatment comprises delivering to the cells a polynucleotide sequence encoding at least one gap junction subunit. In some embodiments, the at least one gap junction subunit is a wild type or mutant connexin. In some embodiments, the gap junction t is chosen from the group consisting of wild type or mutant connexin 43, connexin 30, and connexin 26. In other embodiments, the gap on subunit is conneXin 30.3, in 3l, in 3l.l, in 32, connexin 33, connexin 37, connexin 40, connexin 45, connexin 46 and connexin 50. In some embodiments, the gap junction subunit is modiﬁed to prevent posttranslational modiﬁcations. In some embodiments, the GJIC-increasing treatment comprises delivering to the cells a polynucleotide sequence encoding E-cadherin.

In some embodiments, a GJIC-increasing treatment comprises delivery of a compound to a subject. In some embodiments, the GJIC-increasing treatment comprises delivering to the subject a compound from the group comprising: gemcitabine; cAMP; a retinoic acid; a carotenoid; a glucocorticoid, a ﬂavanoid, apigenin, and/or lovastatin.

In some embodiments, the GJIC-increasing treatment comprises proteasome inhibition. In some embodiments, the GJIC-increasing treatment ses proteasome inhibition by administration ofN—Acetyl-Leu-Leu-Nle-CHO (ALLN) and/or chloroquine.

In some ments, the GJIC-increasing treatment comprises radiation treatment.

In some embodiments, the GJIC-increasing ent comprises electrical ent.

Methods of Detection Disclosed herein, in some embodiments, is a method of measuring the HSV-TK- mediated bystander , the method comprising: a) transfecting cells with a polynucleotide sequence encoding HSV-TK and a ﬁrst ﬂuorescent protein; b) transfecting cells with a second polynucleotide sequence encoding a second ﬂuorescent protein that is optically discernible from the ﬁrst ﬂuorescent protein; c) treating the cells with titrated doses of gancyclovir; and d) measuring the relative amount of expression of the ﬁrst ﬂuorescent protein and the second ﬂuorescent protein.

In one embodiment, red ﬂuorescent proteins (RFPs) are used to quantitate the number of target tumor cells transduced with both the ﬁrst cent protein ﬂuorescent n and the second and Hygro® can be used to select a population of tumor cells in which all express both Hygro® and HSV-TK. RFPs are commercially ble and are contemplated for use herein (see, for e, RFPs described in literature references l-l4 below.

In another embodiment, green ﬂuorescent proteins (GFP) are used to tate the number of transduced target tumor cells. GFPs are are commercially available and are contemplated for use herein including, but not limited to, enhanced green ﬂuorescent n (EGFP).

PLASMIDS AND PRODUCTION OF HSV-TK In some embodiments, disclosed herein are, nucleic acid molecules encoding HSV-TK, or mutants and/or derivatives thereof, which are operably linked to suitable transcriptional or translational regulatory elements. In some embodiments, suitable regulatory elements are derived from bacterial, fungal, viral, mammalian, insect, or plant genes. Selection of appropriate regulatory elements is dependent on the chosen host cell and, in some embodiments, includes: a transcriptional promoter and enhancer or RNA polymerase g sequence, and a ribosomal binding sequence, including a translation initiation signal. bed herein are plasmids, comprising a nucleic acid sequence encoding HSV-TK, or a mutant and/or variant thereof, as described above. In some embodiments, disclosed herein are plasmids encoding HSV-TK fused to a second peptidic component. In some embodiments, the second peptidic component is a eutic agent or polypeptide. In some embodiments, the second ic component is a diagnostic polypeptide.

] In some embodiments, disclosed herein is a y of both viral and non-viral vectors suitable for directing the expression of the nucleic acid molecules encoding HSV-TK disclosed herein.

] In some embodiments, disclosed herein are ds for transfecting and producing delivery vectors or therapeutic vectors for use in therapeutic and diagnostic procedures. In l, such plasmids provide nucleic acid sequences that encode components, viral or non-viral, of targeted vectors disclosed herein. Such plasmids e nucleic acid sequences that encode, for example, the MoMLV pe protein. In some embodiments, the 2014/029814 MOMLV envelope protein is modiﬁed to contain a collagen binding domain. Additional plasmids can include a nucleic acid sequence operably linked to a promoter. The ce generally encodes a Viral gag-pol polypeptide. The d further includes a nucleic acid sequence operably linked to a er, and the sequence encodes a polypeptide that confers drug resistance on the producer cell. An origin of replication is also included. In some embodiments, additional ds comprise an improved HSV-TK encoding sequence, as disclosed herein, 5 ’ and 3 ’ long terminal repeat ces; a ‘I’ retroviral packaging ce, a CMV enhancer upstream of the 5’ LTR promoter, a nucleic acid sequence operably linked to a promoter, and an SV40 origin of replication.

In some embodiments, the polynucleotide encoding HSV-TK is under the control of a le promoter. Suitable promoters include, but are not limited to, the retroviral LTR; the SV40 promoter; the cytomegalovirus (CMV) er; the Rous Sarcoma Virus (RSV) promoter; the histone promoter; the polIII er, the B-actin promoter; inducible promoters, such as the MMTV promoter, the metallothionein promoter; heat shock promoters; adenovirus promoters; the albumin promoter; the ApoAI promoter; B19 irus promoters; human globin promoters; Viral thymidine kinase promoters, such as the Herpes Simplex Virus thymidine kinase promoter; retroviral LTRs; human growth e ers, and the MXIFN inducible promoter. In some embodiments, the promoter is a tissue-speciﬁc promoter. In some embodiments, a tissue speciﬁc promoters is chosen from the group including the nase related promoters (TRP-l and TRP-2), DF3 enhancer (for breast cells), SLPI promoter (secretory leucoprotease inhibitor--expressed in many types of carcinomas), TRS (tissue speciﬁc regulatory sequences), u-fetoprotein promoters (speciﬁc for normal hepatocytes and transformed hepatocytes, respectively), the carcino-embryonic antigen promoter (for use in transformed cells of the intestinal tract, lung, breast and other tissues), the tyrosine hydroxylase promoter (for melanocytes), choline acetyl transferase or neuron speciﬁc enolase promoters for use in lastomas, the regulatory sequence for glial ﬁbroblastomas, the tyrosine hydroxylase promoter, c-erb B-2 promoter, PGK promoter, PEPCK promoter, whey acidic promoter (breast tissue), and casein promoter t ) and the adipocyte P2 promoter. In some embodiments, the promoter is a Viral-speciﬁc promoter (6.g. , retroviral promoters, as well as others such as HIV promoters), hepatitis, herpes (e.g., EBV). In some embodiments, the promoter is the native HSV-TK promoter. In some embodiments, the promoter is a bacterial, fungal or parasitic (e.g. , malarial) -speciﬁc promoter is utilized in order to target a speciﬁc cell or tissue which is infected with a Virus, ia, fungus or parasite.

In some embodiments, the delivery vectors or therapeutic vectors may include a targeting moiety that targets the delivery vectors or therapeutic vectors to a desired cell or system. In some embodiments, the targeting moiety refers to a ligand expressed by the delivery vector or therapeutic vector that is associated with the delivery vehicle and target the e to a cell or . In some embodiments, the ligand may include, but is not limited to, antibodies, receptors and proteins that bind to ar components exposed in or on the ed cell or system. In some embodiments, the exposed ar components may include collagen. In some embodiments, the ligand binding to exposed cellular components comprises proteins that include a collagen binding domain.

The plasmids disclosed herein may be produced by genetic engineering techniques known to those skilled in the art. In addition, the plasmids may be readily expressed by a wide variety of prokaryotic and eukaryotic host cells, including bacterial, mammalian, yeast or other fungi, viral, insect, or plant cells. s for transforming or transfecting such cells to s foreign DNA are well known in the art (see, e.g., Itakura et al., US. Pat. No. 4,704,362; Hinnen et al., PNAS USA 75:1929-1933, 1978; Murray et al., US. Pat. No. 4,801,542; l et al., US. Pat. No. 4,935,349; Hagen et al., US. Pat. No. 4,784,950; Axel et al., US. Pat. No. 4,399,216; Goeddel et al., US. Pat. No. 4,766,075; and Sambrook et al. Molecular g. A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989; for plant cells see Czako and Marton, Plant l. 104: 071, 1994; and Paszkowski et al., Biotech. 24:387-392, 1992).

Protocols for the transfection of mammalian cells are well known to those of ordinary skill in the art. Representative methods include calcium and/or magnesium phosphate mediated transfection, electroporation, lipofection, retroviral, lentiviral ,adenoviral and protoplast fusion-mediated.

In some embodiments, HSV-TK, or a mutant thereof, is prepared by ing the ector systems described above, in order to express the recombinant thymidine kinase mutants. inantly produced thymidine kinase mutants may be ﬁarther purified according to s well known in the art.

In some embodiments, the nucleic acid molecules described herein are introduced into a wide variety of host cells. Representative examples of such host cells include plant cells, eukaryotic cells, and prokaryotic cells. In some embodiments, the nucleic acid molecules are introduced into cells from a vertebrate or warm-blooded animal, such as a human, macaque, dog, cow, horse, pig, sheep, rat, hamster, mouse or fish cell, or any hybrid thereof In some ments, the nucleic acid molecules described herein are introduced into a mammalian cell. In some embodiments, the mammalian cell is chosen from the group including COS, BHK, CHO, HeLa, 293 and NS-1 cells. In some embodiments, suitable expression vectors for directing expression in mammalian cells include a promoter, as well as other transcriptional and translational control sequences. Common promoters include SV40, MMTV, metallothionein-l, adenovirus Ela, Cytomegalovirus Immediate Early Promoter, and the Cytomegalovirus Immediate Late Promoter.

In some embodiments, the nucleic acid molecules described herein are introduced into a yeast or ﬁangi cell. Yeast and ﬁangi host cells suitable for carrying out the present invention include, among others Saccharomyces pombe, Saccharomyces cerevisiae, the genera Pichz’a or Klayveromyces and various species of the genus Aspergillas. le expression vectors for yeast and fungi include, among others, YCp 50 for yeast, and the amdS cloning vector pV3. In some embodiments, transformation of yeast is accomplished either by preparation of spheroplasts of yeast with DNA or by ent with alkaline salts such as LiCl.

In some embodiments, transformation of ﬁangi is d out using polyethylene .

In some embodiments, the nucleic acid les described herein are uced into a bacterial cell. Bacterial host cells suitable for carrying out the present invention include E. coli, B. sabtz’lz’s, Salmonella typhz'marz’am, and various s within the genus' Pseudomonas, Streptomyces, and Staphylococcus, as well as many other bacterial species well known to one of ordinary skill in the art. Representative examples of bacterial host cells include DH50L (Stratagene, La Jolla, Calif).

In some embodiments, ial expression vectors comprise a promoter which functions in the host cell, one or more selectable phenotypic markers, and a bacterial origin of replication. Representative promoters include the B-lactamase (penicillinase) and lactose promoter system, the T7 RNA polymerase promoter, the lambda promoter, the trp promoter and the tac promoter. Representative selectable markers include various antibiotic ance markers such as the kanamycin or ampicillin resistance genes. In some embodiments, plasmids suitable for orming host bacterial cells include, among others, pBR322, the pUC plasmids pUC l 8, pUCl9, pUCl 18, pUCl l9, pNH8A, pNHl6a, pNH18a, and Bluescript M13 (Stratagene, La Jolla, Calif.).

In some embodiments, the nucleic acid molecules described herein are expressed in non-human transgenic animals such as mice, rats, rabbits, sheep, dogs and pigs. In some embodiments, an expression unit, including a nucleic acid molecule to be expressed together with appropriately positioned expression control sequences, is introduced into pronuclei of fertilized eggs, for example, by microinjection. In some embodiments, integration of the injected DNA is ed by blot is ofDNA from tissue samples. In some embodiments, the uced DNA is orated into the germ line of the animal so that it is passed on to the 's progeny. In some embodiments, tissue-speciﬁc expression is ed through the use of a tissue-speciﬁc promoter, or through the use of an inducible promoter, such as the othionein gene promoter, which allows regulated expression of the transgene.

In some embodiments, the nucleic acid molecules described herein are introduced into host cells by a wide variety of mechanisms, including for example calcium phosphate- mediated ection; lipofection; gene gun; electroporation; retroviral, adenoviral, protoplast fusion-mediated transfection or DEAE-dextran mediated transfection.

VECTORS AND METHODS OF TION THEREOF ] Disclosed herein is a vector particle, comprising an improved HSV-TK encoding sequence, as described above, which is to be expressed in a desired cell. In some embodiments, the vector particle is a viral vector particle. In some embodiments, the viral vector particle is a retroviral vector particle.

In some embodiments, a vector le comprising an improved HSV-TK encoding sequence contains or expresses a wide variety of additional nucleic acid molecules in addition to the improved HSV-TK encoding sequence. In some embodiments, the vector additionally expresses a lymphokine, antisense sequence, toxin or “replacement” protein (e.g., adenosine deaminase). entative examples of lymphokines e, for example, IL-l, IL- 2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-lO, IL-l l, IL-12, IL-l3, IL-l4, IL-l5, GM-CSF, G-CSF, M-CSF, alpha-interferon, nterferon, gamma interferon, and tumor necrosis factors (TNFs). Representative es of antisense sequences include, but are not limited to: antisense myc, antisense p53, antisense ms, as well as antisense sequences which block the expression or production of viruses such as HIV, HBV and HCV. Representative examples of toxins include, but are not limited to: ricin, abrin, diphtheria toxin, cholera toxin, gelonin, botulinum, pokeweed antiviral protein, tritin, Shigella toxin, and monas exotoxin A.

Representative examples of suicide genes e, but are not limited to: a cytosine deaminase, a VSV-tk, IL-2, nitroreductase (NR), ylesterase, beta-glucuronidase, rome p450, beta-galactosidase, diphtheria toxin A-chain (DT-A), carboxypeptide G2 (CPG2), purine nucleoside phosphorylase (PNP), and deoxycytidine kinase (dCK). In some instances, the vector additionally expresses a yeast and/or a bacterial cytosine deaminase.

Additional eutic sequences include, but are not limited to, Yeast or ial Cytosine Deaminase, other suicide genes and other apoptotic genes, guanylate , p53 kinase, IL-12 and other immune stimulatory or cytokine genes, GFP, RFP, iRFP, LUC2, GLUC and other ﬂuorescent and bioluminescent genes, Cyclin A, D and other cell cycle regulatory genes, Viral genes, ial genes, human genes, synthetic genes, SIRNA, RNAi, Micro RNA, antisense of genes, inhibitory or stimulatory sequences, genes captured from library strategies, repeat sequence, replication sequence, promoter or enhancer sequence, DNA binding ces, any therapeutic sequence, etc.

In some embodiments, a polynucleotide sequence encoding a or to a gamma retrovirus is included. Disclosed herein in the present application are experiments demonstrating that that the receptor binding domain (RBD) of amphotropic viral vector envelope gene product binds to a PiT-2 receptor on the cell membrane of target cells and allows for enhancement of viral vector transduction. Using a topological model for PiT-2 and a murine leukemia virus (A-MuLV) receptor-binding assay on CHO-Kl and BHK cells, Feldman et al.

(Eiden MV. J Virol. (2004) 78: 595—602) identiﬁed the extracellular domain one (ECDl) of the human PiT-2 receptor as being important for amphotrophic virus g and infection. Studies by Bottger and Petersen (2004) showed that the part needed for binding the virus could be narrowed down to the 182 aa N-Term region and 170 aa C-Term region.

Accordingly, also provided herein in select embodiments are polynucleotide sequences encoding a mutated form of thymidine kinase from human simplex virus (HSV-TK), wherein the encoded HSV-TK includes a polynucleotide sequence to encode PiT-2, PiT-l, MCAT and other ors used by gamma retrovirus.

GAP JUNCTION INTRACELLULAR COMMUNICATION In some embodiments, a vector particle additionally ses a gap junction intracellular communication (GJIC)-increasing treatment, as bed . In some embodiments, a vector le additionally expresses one or more genes which encode proteins that facilitate or increase the biological activity of ine kinase. In some embodiments, a vector further comprises a sequence encoding a DNA polymerase (e.g., a Herpes DNA polymerase) and/or guanylate kinase.

One of the most frequently used delivery systems for achieving gene therapy involves viral s, most ly adenoviral and retroviral vectors. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; US. Pat. No. 5,219,740; WO 30; WO 93/10218; US. Pat. No. 4,777,127; GB Patent No. 2,200,651; EP 0 345 242; and WO 91/02805; each of which is incorporated by reference with t to the disclosures regarding recombinant retroviruses), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR—373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and adeno-associated virus (AAV) s (see, e. g., W0 94/12649, WO 93/03769; W0 W0 2014/153258 93/19191; W0 94/28938; W0 84 and W0 95/00655). Administration ofDNA linked to killed adenovirus as described by Curiel (Hum. Gene Ther. (1992) 3:147) can also be employed.

Retroviruses generally have three common open reading frames, gag, pol, and env, which encode the matrix, gag and nucleocapsid structural proteins, encode enzymes including e transcriptase, integrase and protease, and encode envelope proteins and transmembrane fusogenic proteins, tively. Generally, retroviral vector particles are ed by packaging cell lines that provide the necessary gag, pol, and env gene products in trans. This approach results in the production of retroviral vector les which transduce mammalian cells, but are incapable of ﬁlrther replication after they have integrated into the genome of the cell.

For gene delivery purposes, a viral particle can be developed from a virus that is native to a target cell or from a virus that is non-native to a target cell. Generally, it is desirable to use a non-native virus vector rather than a native virus vector. While native virus vectors may possess a natural y for target cells, such viruses pose a greater hazard since they possess a greater potential for propagation in target cells. In this regard, animal virus vectors, wherein they are not naturally designed for propagation in human cells, can be useful for gene delivery to human cells. In order to obtain sufﬁcient yields of such animal virus s for use in gene delivery, however, it is necessary to carry out tion in a native animal packaging cell.

Virus vectors produced in this way, however, normally lack any components either as part of the envelope or as part of the capsid that can provide m for human cells. For e, current practices for the production of non-human virus vectors, such as ecotropic mouse (murine) retroviruses like MMLV, are produced in a mouse packaging cell line. Another component required for human cell tropism must be provided.

In general, the ation of a viral vector (without a helper virus) proceeds in a packaging cell in which nucleic acid sequences for packaging components are stably integrated into the cellular genome and nucleic acid coding for viral nucleic acid is introduced in such a cell line.

] In some embodiments, the retroviral plasmid vector includes a polynucleotide comprising the improved HSV-TK encoding sequence, and the expression vehicle including the polynucleotide comprising the improved HSV-TK encoding ce are transduced into a packaging cell line including nucleic acid sequences encoding the gag, pol, and wild-type (2'.e., unmodiﬁed) env retroviral proteins. Examples of such ing cell lines include, but are not limited to, the PESOl, PA3 l7 (ATCC No. CRL 9078),'-2,—AM, PAlZ, Tl9-l4X, VT-l9-l7-H2, TCRE, TCRIP, GP+E-86, GP+envAm12, and DAN cell lines as described in Miller, Human Gene Therap, Vol. 1, pgs. 5-14 (1990), which is orated herein by reference in its entirety, or the 293T cell line (U. S. Patent No. 5,952,225). The vector(s) may be transfected into the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, and use of liposomes, such as hereinabove described, and CaPO4 precipitation.

Such producer cells generally generate infectious retroviral vector particles which include the ﬁrst, or unmodiﬁed wild-type retroviral envelope protein, a chimeric retroviral envelope protein, and a polynucleotide encoding the therapeutic or diagnostic agent.

In some embodiments, there is provided a packaging cell which includes polynucleotides encoding the gag and pol proteins, a polynucleotide encoding a ﬁrst retroviral envelope protein free of non-retroviral peptides (which, in some embodiments, is a ype retroviral envelope protein), and a polynucleotide encoding a chimeric retroviral envelope protein. In some embodiments, a producer cell for generating retroviral vector particles which e the ﬁrst and chimeric envelope proteins is ed by introducing into such packaging cell either a iral vector particle or a retroviral d vector, in each case including a polynucleotide encoding the eutic or diagnostic agent. In some ments, the producer cell line thus generates infectious retroviral vector particles including the polynucleotide comprising the ed HSV-TK encoding sequence.

In some embodiments, disclosed herein is a kit for the production of viral vectors, the kit comprising: a) a container containing a ﬁrst plasmid comprising a nucleic acid ce encoding a iral envelope protein, wherein the c acid sequence is operably linked to a promoter; b) a container containing a second d comprising: a nucleic acid sequence operably linked to a promoter, wherein the sequence encodes a viral gag-pol polypeptide, a nucleic acid sequence operably linked to a promoter, wherein the sequence encodes a polypeptide that confers drug resistance on the producer cell, and an SV40 origin of replication; c) a container containing a third plasmid comprising: an improved HSV-TK ng sequence operably linked to a er, 5 ’ and 3 ’ long terminal repeat sequences (LTRs), a ‘I’ retroviral packaging sequence, a CMV promoter upstream of the 5’ LTR; a nucleic acid sequence operably linked to a promoter, wherein the ce encodes a polypeptide that confers drug resistance on the producer cell, an SV40 origin of replication, d) a container containing a producer cell that expresses SV40 large T n; and e) instructions for transiently transfecting the er cell of d) with the plasmids of a), b), and c) and culturing the transfected producer cell under conditions that allow viral les to be produced.

It is recognized that the delivery vectors or therapeutic vectors disclosed herein include viral and non-viral particles. Non-viral delivery systems, such as microparticles or nanoparticles including, for e, cationic liposomes and polycations, provide alternative methods for delivery systems and are encompassed by the present disclosure. Non-viral WO 53258 particles include encapsulated proteins, including wholly or partially assembled viral particles, in lipid bilayers. Methods for encapsulating viruses into lipid bilayers are known in the art. They include passive entrapment into lipid bilayer-enclosed vesicles (liposomes), and incubation of virions with liposomes (US. Pat. No. 5,962,429; Fasbender, et al., J. Biol. Chem. 79-6489; Hodgson and Solaiman, Nature Biotechnology -342 (1996)). Without being limited by a theory, we assume that acidic proteins exposed on the surface of a virion provide an interface for complexation with the ic cationic polymer component of the delivery vector or therapeutic vector and serve as a “scaffold” for the bilayer formation by the neutral lipid component.

Examples of non-viral delivery systems include, for example, Wheeler et al., US.

Pat. Nos. 5,976,567 and 5,981,501. These s disclose preparation of serum-stable plasmid- lipid particles by contacting an aqueous solution of a plasmid with an c solution containing cationic and non-cationic lipids. Thierry et al., US. Pat. No. 6,096,335 disclose preparation of a complex comprising a ly c biologically active substance, a cationic constituent, and an anionic constituent. Allen and Stuart, 98/12937 (WO 98/5 8630) disclose forming polynucleotide-cationic lipid particles in a lipid solvent suitable for solubilization of the cationic lipid, adding neutral vesicle-forming lipid to the t containing the particles, and evaporating the lipid solvent to form liposomes having the polynucleotide entrapped within. Allen and Stuart, US. Pat. No. 6,120,798, disclose forming polynucleotide- lipid microparticles by dissolving a polynucleotide in a ﬁrst, e. g., aqueous, solvent, dissolving a lipid in a , e.g., c, t immiscible with said ﬁrst solvent, adding a third solvent to effect formation of a single phase, and r adding an amount of the ﬁrst and second solvents to effect formation of two liquid phases. Bally et al. US. Pat. No. 5,705,385, and Zhang et al. US. Pat. No. 6,110,745 disclose a method for preparing a lipid-nucleic acid particle by contacting a nucleic acid with a solution containing a non-cationic lipid and a cationic lipid to form a nucleic acid mixture. Maurer et al., PCT/CA00/00843 (WO 01/06574) disclose a method for preparing ﬁllly lipid-encapsulated therapeutic agent particles of a charged therapeutic agent including combining preformed lipid vesicles, a charged therapeutic agent, and a destabilizing agent to form a mixture thereof in a destabilizing solvent that destabilizes, but does not disrupt, the vesicles, and subsequently removing the destabilizing agent.

A Particle-Forming Component (“PFC”) typically comprises a lipid, such as a ic lipid, optionally in combination with a PFC other than a cationic lipid. A cationic lipid is a lipid whose molecule is capable of electrolytic dissociation producing net positive ionic charge in the range ofpH from about 3 to about 10, preferably in the physiological pH range from about 4 to about 9. Such cationic lipids encompass, for e, cationic detergents such as cationic amphiphiles having a single hydrocarbon chain. Patent and scientiﬁc literature describes numerous cationic lipids having nucleic acid transfection-enhancing ties. These transfection-enhancing cationic lipids include, for example: 1,2-dioleyloxy(N,N,N— hylammonio)propane chloride-, DOTMA (US. Pat. No. 4,897,355); DOSPA (see Hawley-Nelson, et al., Focus 15(3):73 (1993)); N,N-distearyl-N,N-dimethyl-ammonium bromide, or DDAB (US. Pat. No. 5,279,833); 1,2-dioleoyloxy(N,N,N-trimethylammonio) propane chloride-DOTAP tatos, et al., Biochemistry 27: 3917-3925 ); glycerol based lipids (see Leventis, et al., Biochem. Biophys. Acta 1023:124 (1990); l-PE (US.

Pat. No. 5,980,935); lysinyl-PE (Puyal, et al. J. Biochem. 228:697 (1995)), lipopolyamines (US.

Pat. No. 5,171,678) and cholesterol based lipids (WO 93/05162, US. Pat. No. 5,283,185); CHIM (1-(3-cholesteryl)-oxycarbonyl-aminomethylimidazole); and the like. Cationic lipids for transfection are reviewed, for example, in: Behr, Bioconjugate Chemistry, 5 :3 82-3 89 (1994).

Preferable ic lipids are DDAB, CHIM, or combinations thereof. Examples of cationic lipids that are cationic detergents include (C12-C18)-alkyl- and (C 12-C18)-alkenyl- trimethylammonium salts, N-(C12-C18)-alkyl- and 2-C18)-alkenyl-pyridinium salts, and the like.

In some embodiments, the size of a delivery vector or therapeutic vector formed is within the range of about 40 to about 1500 nm. In some ments, the delivery vector or therapeutic vector is in the range of about 50-500 nm in size. In some embodiments, the delivery vector or eutic vector is in the range of about 20-150 nm in size. This size selection advantageously aids the delivery vector, when it is administered to the body, to penetrate from the blood vessels into the diseased tissues such as malignant tumors, and transfer a therapeutic nucleic acid therein. It is also a characteristic and advantageous property of the ry vector that its size, as measured for example, by dynamic light ring method, does not substantially increase in the presence of ellular biological ﬂuids such as in Vitro cell culture media or blood plasma.

Alternatively, in some embodiments, cells which produce retroviruses are injected into a tumor. In some embodiments, the retrovirus-producing cells so introduced are engineered to ly produce a delivery vector, such as a Viral vector particle, so that continuous productions of the vector ed within the tumor mass in situ. In some embodiments, proliferating tumor cells are transduced in vivo by proximity to retroviral vector- producing cells.

METHODS OF USE In some embodiments, disclosed herein is a method of providing to target cells a cleotide encoding HSV-TK, as disclosed herein, the method comprising and then ng the cells to an appropriate substrate which is converted to a toxic substance to kill those cells expressing the mutant HSV-l thymidine kinase gene as well as those in the vicinity of the mutant HSV-l thymidine kinase gene-expressing cells, z'.e. cells. The mutant , bystander HSV-l thymidine kinase gene can be administered directly to the targeted or d cells or systemically in ation with a targeting means, such as through the ion of a particular viral vector or delivery formulation. Cells can be treated in viva, within the patient to be treated, or treated in vitro, then injected into the patient. Following introduction of the mutant HSV-l thymidine kinase gene into cells in the patient, the prodrug is administered, systemically or y, in an effective amount to be converted by the mutant HSV-l thymidine kinase into a ient amount of toxic substance to kill the targeted cells. A nucleoside analog which is a substrate for HSV-l TK to e a toxic substance which kills target cells is referred to herein as a “prodrug”.

In some embodiments, disclosed herein is a method of killing a cell, the method comprising: i) introducing into the cell a polynucleotide or vector as disclosed herein; ii) allowing or directing the cell to express thymidine kinase; and iii) contacting the cell with an agent that is converted by thymidine kinase to a cytotoxic agent.

In some embodiments of the present invention there is provided herein a method of preventing graft-versus-host disease (GvHD) in a patient comprising: (i) administering to a host T-cells genetically engineered to include a polynucleotide or vector of the present invention; and (ii) administering to said host, prior to the occurrence of graft-versus-host disease, an agent capable of being converted by thymidine kinase to a cytotoxic agent in an amount effective to kill genetically ered T-cells capable of effecting GvHD. During an allogeneic bone marrow transplant, alloreactive T lymphocytes can be removed from the graft in order to prevent graft versus host disease. GvHD occurs when T-cells in the lanted stem cell graft attack the transplant recipient's body. However, l of the s can increase the incidence of disease relapse, graft rejection and reactivation of viral infection. To counter the possibility of GvHD, allogeneic bone marrow transplant patients can be treated by introducing donor T lymphocytes after a delay following the allogeneic bone marrow transplant. However, delayed introduction of donor T lymphocytes following allogeneic bone marrow transplant is limited by GvHD, a nt and potentially lethal complication of the treatment. By stering to a transplant recipient T-cells genetically engineered to e a polynucleotide encoding a “suicide gene,” the T-cells can be killed if they begin to attack the transplant ent’s body.

In some embodiments, the iral vector particles, which include a chimeric retroviral envelope protein and a cleotide encoding a therapeutic agent, are stered to a host in order to express the therapeutic agent in the host. In some embodiments, the polynucleotide encoding a therapeutic agent is a polynucleotide encoding HSV-TK, or a mutant and/or variant thereof, as disclosed herein.

In some embodiments, cells are obtained from a patient, and retroviral vector particles are used to introduce a therapeutic agent or polypeptide into the cells, and such modiﬁed cells are administered to the patient. In some embodiments, retroviral vector particles are administered to the patient in viva, whereby the retroviral vector particles transduce cells of the patient in viva.

In some ments, disclosed herein is a method of delivering a therapeutic agent or ptide to a site of tissue injury in a subject, comprising directly or enously delivering to the site of tissue injury a iral particle comprising: i) a chimeric retroviral envelope protein and ii) at least one polynucleotide encoding a therapeutic ptide, wherein the viral particle binds to en exposed at the site of tissue injury and expresses the therapeutic polypeptide at the site of tissue injury. In some embodiments, the tissue injury is selected from the group consisting of tissue injury due to tumor invasion, vascular lesion, ulcerative lesions, atory tissue injury, laser injury to eyes, surgery, arthritic joints, scars, and keloids. In some embodiments, the tissue injury is a lesion of tissue due to growth of a tumor in the host.

In some embodiments, therapeutic vectors, as disclosed herein, are employed in the treatment of cancer, including malignant and nonmalignant tumors. In some ments, the therapeutic vectors ﬁlrther comprise an extracellular matrix binding e or peptide domain. In some embodiments, the extracellular matrix binding peptide or peptide domain is a collagen binding domain or peptide. In some embodiments, the tumors include, but are not limited to, all solid tumors.

In some embodiments, eutic vectors, as sed , are employed in the treatment of cancer being selected from the group consisting of breast cancer, skin , bone cancer, prostate cancer, liver cancer, lung cancer, brain cancer, cancer of the larynx, gall bladder, pancreas, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma of both ulcerating and papillary type, metastatic skin carcinoma, melanoma, osteosarcoma, Ewing’s a, veticulum cell sarcoma, myeloma, giant cell tumor, small-cell lung tumor, gallstones, islet cell WO 53258 tumor, primary brain tumor, acute and chronic cytic and granulocytic tumors, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neurons, intestinal ganglloneuromas, hyperplastic corneal nerve tumor, marfanoid s tumor, Wilm’s tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia and in situ carcinoma, lastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosis ﬁJngoide, myosarcoma, ’s sarcoma, osteogenic and other sarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera, adenocarcinoma, glioblastoma multiforma, leukemias, lymphomas, ant melanomas, and epidermoid carcinomas. In other embodiments, the cancer being treated is pancreatic cancer, liver cancer, breast cancer, osteosarcoma, lung cancer, soft tissue sarcoma, cancer of the larynx, melanoma, n cancer, brain cancer, Ewing’s sarcoma or colon cancer.

In other embodiments, the cancer to be treated is chosen from the group consisting of primary hepatocellular carcinoma, metastatic breast carcinoma to liver, metastatic pancreatic cancer to liver, metastatic gastric cancer to liver, metastatic esophageal cancer to liver, metastatic lung cancer to liver, metastatic melanoma to liver, metastatic ovarian oma to liver and metastatic kidney cancer to liver.

The therapeutic vectors may be stered alone or in conjunction with other therapeutic treatments or active agents. Examples of other active agents that may be used include, but are not limited to, chemotherapeutic agents, nﬂammatory agents, protease inhibitors, such as HIV protease inhibitors, nucleoside analogs, such as AZT. In some embodiments, the methods of treatment ﬁarther comprise administering to the subject a chemotherapeutic agent, a biologic agent, or radiotherapy prior to, contemporaneously with, or subsequent to the administration of the eutic Viral particles. One of skill in the art will appreciate that the retroviral particles described herein may be administered either by the same route as the one or more agents (6.g. the retroviral vector and the agent are both administered intravenously) or by ent routes (6.g. , the retroviral vector is administered intravenously and the one or more agents are administered orally).

The dosage of the therapeutic Viral particles lies preferably within a range of circulating concentrations that e the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. A eutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a ating plasma concentration range that es the IC50 (z'.e., the concentration of the test compound which achieves a half- maximal infection or a half-maximal inhibition) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by RT-qPCR or ddPCR methods.

An effective amount or therapeutically effective of the retroviral particles disclosed herein to be administered to a t in need of treatment may be determined in a y of ways. By way of example, the amount may be based on viral titer or efficacy in an animal model. Alternatively the dosing regimes used in clinical trials may be used as general guidelines.

In some embodiments, the daily dose may be administered in a single dose or in portions at various hours of the day. In some embodiments, a higher dosage may be required and may be reduced over time when the optimal initial response is obtained. In some embodiments, treatment may be continuous for days, weeks, or years, or may be at intervals with intervening rest periods. In some embodiments, the dosage is modified in accordance with other treatments the individual may be receiving. However, the method of ent is in no way limited to a particular concentration or range of the retroviral particle and may be varied for each individual being treated and for each tive used.

Individualization of dosage may be required to e the maximum effect for a given individual. In some embodiments, the dosage administered to an individual being treated varies depending on the individual’s age, severity or stage of the disease and response to the course of treatment. In some embodiments, clinical parameters for ining dosage include, but are not d to, tumor size, alteration in the level of tumor markers used in clinical testing for particular malignancies. In some embodiments, the ng physician determines the therapeutically effective amount to be used for a given dual. In some embodiments, the therapies disclosed herein are administered as often as necessary and for the period of time judged necessary by the treating physician.

The therapeutic vectors, including but not limited to the therapeutic retroviral particles that are specifically to the cell or system of st, may be systemically or regionally (locally) delivered to a subject in need of treatment. For e, the therapeutic vectors may be systemically administered intravenously. Alternatively, the therapeutic vectors may also be administered intra-arterially. The therapeutic s may also be administered topically, intravenously, intra-arterially, intra-tumorally, intracolonically, intratracheally, intraperitoneally, intranasally, intravascularly, intrathecally, intracranially, intramarrowly, intrapleurally, intradermally, subcutaneously, intramuscularly, cularly, intraosseously and/or intrasynovially or sterotactically. A combination of delivery modes may also be used, for e, a t may e the eutic vectors both systemically and regionally (locally) to improve tumor responses with treatment of the therapeutic vectors.

In some embodiments, multiple therapeutic s (e.g., ﬁrst and second therapeutic course) are administered to a t in need of treatment. In some embodiments, the ﬁrst and/or second eutic course is stered intravenously. In other embodiments, the ﬁrst and/or second therapeutic course is stered Via intra-arterial infusion, including but not limited to infusion through the hepatic artery, cerebral artery, coronary artery, pulmonary artery, iliac artery, celiac trunk, gastric artery, splenic , renal artery, gonadal artery, subclaVian artery, vertebral artery, axilary artery, brachial artery, radial artery, ulnar artery, carotid artery, femoral artery, inferior mesenteric artery and/or superior mesenteric artery. Intra- arterial infusion may be accomplished using endovascular procedures, percutaneous ures or open surgical approaches. In some embodiments, the ﬁrst and second therapeutic course may be stered sequentially. In yet other embodiments, the ﬁrst and second therapeutic course may be administered simultaneously. In still other embodiments, the optional third therapeutic course may be administered sequentially or simultaneously with the ﬁrst and second eutic courses.

In some embodiments, the therapeutic vectors disclosed herein may be administered in conjunction with a sequential or concurrently administered therapeutic course(s) in high doses on a cumulative basis. For e, in some embodiments, a patient in need thereofmay be systemically administered, e.g., intravenously administered, with a ﬁrst therapeutic course of at least I x 109 TVP, at least I x 1010 TVP, at least I x 1011 TVP, at least I x 1012 TVP, at least I x 1013 TVP, at least I x 1014 TVP, at least I x 1015 TVP, at least I x 1016 TVP, at least I x 1017 TVP, at least I x 1018 TVP, at least I x 1019 TVP, at least I x 1020 TVP, at least I x 1021 TVP or at least I x 1022 TVP delivery vector on a cumulative basis. The ﬁrst therapeutic course may be systemically administered. Alternatively, the ﬁrst therapeutic course may be administered in a localized manner, e.g., intra-arterially, for example a patient in need thereofmay be administered Via intra-arterial infusion with at least of at least I x 109 TVP, at least I x 1010 TVP, at least I x 1011 TVP, at least I x 1012 TVP, at least I x 1013 TVP, at least I x 1014 TVP, at least I x 1015 TVP, at least I x 1016 TVP, at least I x 1017 TVP, at least I x 1018 TVP, at least I x 1019 TVP, at least I x 1020 TVP, at least I x 1021 TVP or at least I x 1022 TVP delivery vector on a cumulative basis.

In yet other embodiments, a t in need thereofmay receive a ation, either sequentially or concurrently, of systemic and intra-arterial infusions administration of high doses of delivery vector. For example, a patient in need thereofmay be ﬁrst systemically administered with at least of at least I x 109 TVP, at least I x 1010 TVP, at least I x 1011 TVP, at least I x 1012 TVP, at least I x 1013 TVP, at least I x 1014 TVP, at least I x 1015, at least I x 1016 TVP, at least I x 1017 TVP, at least I x 1018 TVP, at least I x 1019 TVP, at least I x 1020 TVP, at least 1 x 1021 TVP or at least 1 x 1022 TVP delivery vector on a cumulative basis, followed by an additional therapeutic course of intra-arterial inﬁlsion, e.g., hepatic arterial infusion, administered delivery vector of at least of at least 1 x 109 TVP, at least 1 x 1010 TVP, at least 1 x 1011 TVP, at least 1 x 1012 TVP, at least 1 x 1013 TVP, at least 1 x 1014 TVP, at least 1 x 1015 TVP, at least 1 x 1016 TVP, at least 1 x 1017 TVP, at least 1 x 1018 TVP, at least 1 x 1019 TVP, at least 1 x 1020 TVP, at least 1 x 1021 TVP or at least 1 x 1022 TVP on a cumulative basis. In still another embodiment, a patient in need fmay receive a combination of intra-arterial infusion and systemic administration of delivery vector in high doses. For example, a patient in need thereofmay be ﬁrst be administered via intra-arterial inﬁlsion with at least of at least 1 x 109 TVP, at least 1 x 1010 TVP, at least 1 x 1011 TVP, at least 1 x 1012 TVP, at least 1 x 1013 TVP, at least 1 x 1014 TVP, at least 1 x 1015 TVP, at least 1 x 1016 TVP, at least 1 x 1017 TVP, at least 1 x 1018 TVP, at least 1 x 1019 TVP, at least 1 x 1020 TVP, at least 1 x 1021 TVP or at least 1 x 1022 TVP delivery vector on a cumulative basis, ed by an additional therapeutic course of systemically administered delivery vector of at least of at least 1 x 109 TVP, at least 1 x 1010 TVP, at least 1 x 1011 TVP, at least 1 x 1012 TVP, at least 1 x 1013 TVP, at least 1 x 1014 TVP, at least 1 x 1015 TVP, at least 1 x 1016 TVP, at least 1 x 1017 TVP, at least 1 x 1018 TVP, at least 1 x 1019 TVP, at least 1 x 1020 TVP, at least 1 x 1021 TVP or at least 1 x 1022 TVP on a cumulative basis. The therapeutic courses may also be administered simultaneously, i.e., a therapeutic course of high doses of delivery vector, for example, at least of at least 1 x 109 TVP, at least 1 x 1010 TVP, at least 1 x 1011 TVP, at least 1 x 1012 TVP, at least 1 x 1013 TVP, at least 1 x 1014 TVP, at least 1 x 1015 TVP, at least 1 x 1016 TVP, at least 1 x 1017 TVP, at least 1 x 1018 TVP, at least 1 x 1019 TVP, at least 1 x 1020 TVP, at least 1 x 1021 TVP or at least 1 x 1022 TVP ry vector on a cumulative basis, together with a therapeutic course of intra-arterial inﬁlsion, e.g., hepatic arterial on, administered delivery vector of at least of at least 1 x 109 TVP, at least 1 x 1010 TVP, at least 1 x 1011 TVP, at least 1 x 1012 TVP, at least 1 x 1013 TVP, at least 1 x 1014 TVP, at least 1 x 1015 TVP, at least 1 x 1016 TVP, at least 1 x 1017 TVP, at least 1 x 1018 TVP, at least 1 x 1019 TVP, at least 1 x 1020 TVP, at least 1 x 1021 TVP or at least 1 x 1022 TVP on a tive basis.

In still other embodiments, a t in need thereofmay onally e, either sequentially or concurrently with the first and second therapeutic courses, additional therapeutic courses (e.g., third therapeutic course, fourth therapeutic course, fifth therapeutic course) of cumulative dose of delivery vector, for example, at least of at least 1 x 109 TVP, at least 1 x 1010 TVP, at least 1 x 1011 TVP, at least 1 x 1012 TVP, at least 1 x 1013 TVP, at least 1 x 1014 TVP, at least 1 x 1015 TVP, at least 1 x 1016 TVP, at least 1 x 1017 TVP, at least 1 x 1018 TVP, at least I x 1019 TVP, at least I x 1020 TVP, at least I x 1021 TVP or at least I x 1022 TVP delivery vector on a tive basis.

In some embodiments, the subject in need of treatment is administered systemically (e.g., intravenously) a dose of at least I x 1011 TVP, followed by the administration via intra-arterial infusion (e.g., hepatic-arterial infusion) of a dose of at least I x 1011 TVP. In other ments, the patient in need of treatment may be administered ically (e.g., intravenously) a cumulative dose of at least I x 1012 TVP, followed by the administration via intra-arterial inﬁlsion (e.g., hepatic-arterial infusion) of a dose of at least I x 1012 TVP. In one embodiment, the patient in need of treatment may be administered systemically (e.g., intravenously) a dose of at least I x 1013 TVP, followed by the administration via intra-arterial infusion (e.g., hepatic-arterial infusion) of a dose of at least I x 1013 TVP. In yet other embodiments, the patient in need of treatment may be administered systemically (e.g., intravenously) a dose of at least I x 1014 TVP, concurrently with the stration via intra- arterial infusion (e.g., hepatic-arterial infusion) of a dose of at least I x 1014 TVP. In still other embodiments, the t in need of ent may be administered systemically (e.g., intravenously) a dose of at least I x 1015 TVP, together with the administration via intra-arterial on (e.g., hepatic-arterial infusion) of a dose of at least I x 1015 TVP. In yet other embodiments, the patient in need of treatment may be administered systemically (e.g., intravenously) a dose of at least I x 1016 TVP, concurrently with the administration via intra- arterial infusion (e.g., hepatic-arterial infusion) of a dose of at least I x 1016 TVP. In still other ments, the patient in need of treatment may be administered systemically (e.g., intravenously) a dose of at least I x 10137TVP, er with the administration via intra-arterial infusion (e.g., hepatic-arterial infusion) of a dose of at least I x 1017 TVP.

A subject in need of treatment may also be administered, either systemically or localized (for example intra-arterial infusion, such as hepatic arterial inﬁlsion) a eutic course of delivery vector for a deﬁned period of time. In some embodiments, the period of time may be at least one day, at least two days, at least three days, at least four days, at least ﬁve days, at least six days, at least seven days, at least one week, at least two weeks, at least three weeks, at least four weeks, at least ﬁve weeks, at least six weeks, at least seven weeks, at least eight weeks, at least 2 months, at least three months, at least four months, at least ﬁve months, at least six , at least seven months, at least eight , at least nine months, at least ten months, at least eleven months, at least one year, at least two years, at least three years, at least four years, or at least ﬁve years. Administration could also take place in a chronic manner, z'.e., for an undeﬁned or indeﬁnite period of time. stration of the therapeutic vector may also occur in a periodic manner, e.g., at least once a day, at least twice a day, at least three times a day, at least four times a day, at least ﬁve times a day. Periodic administration of the delivery vector may be ent upon the time of delivery vector as well as the mode of administration. For example, parenteral administration may take place only once a day over an extended period of time, whereas oral administration of the delivery vector may take place more than once a day wherein administration of the ry vector takes place over a r period of time.

In one embodiment, the subject is allowed to rest 1 to 2 days between the ﬁrst therapeutic course and second therapeutic . In some embodiments, the subject is allowed to rest 2 to 4 days between the ﬁrst therapeutic course and second therapeutic course. In other embodiments, the subject is allowed to rest at least 2 days n the ﬁrst and second therapeutic course. In yet other embodiments, the subject is allowed to rest at least 4 days between the ﬁrst and second therapeutic course. In still other ments, the subject is allowed to rest at least 6 days between the ﬁrst and second therapeutic course. In some embodiments, the subject is allowed to rest at least 1 week between the ﬁrst and second therapeutic course. In yet other embodiments, the subject is d to rest at least 2 weeks between the ﬁrst and second therapeutic course. In one embodiment, the subject is allowed to rest at least one month n the ﬁrst and second therapeutic course. In some embodiments, the subject is allowed to rest at least 1-7 days between the second therapeutic course and the optional third therapeutic course. In yet other embodiments, the subject is allowed to rest at least 1-2 weeks between the second therapeutic course and the optional third therapeutic course.

In some embodiments, the therapeutic vector is administered to increase local concentration of the peptide or vector. In some embodiments, the therapeutic vector is administered via intra-arterial inﬁlsion, which increases local tration of the therapeutic vector to a speciﬁc organ . In yet other embodiments, the therapeutic vector is administered intra-tumorally. Dependent upon the on of the target lesions, in some embodiments, catheterization of the hepatic artery is followed by on into the pancreaticoduodenal, right hepatic, and middle hepatic artery, respectively, in order to locally target hepatic s. In some embodiments, localized distribution to other organ systems, ing the lung, gastrointestinal, brain, reproductive, splenic or other deﬁned organ , of the peptide or delivery vector is accomplished via catheterization or other localized delivery . In some embodiments, intra-arterial infusions are accomplished via any other available arterial source, including but not limited to infusion through the hepatic artery, cerebral artery, coronary artery, pulmonary artery, iliac artery, celiac trunk, gastric artery, splenic artery, renal artery, gonadal artery, subclavian artery, vertebral artery, axilary artery, brachial artery, radial artery, ulnar artery, carotid artery, femoral artery, inferior eric artery and/or superior mesenteric . In some embodiments, arterial infusion is accomplished using scular procedures, percutaneous procedures or open surgical approaches.

Formulations Pharmaceutical compositions comprising a therapeutic vector can be formulated in any conventional manner by mixing a selected amount of the therapeutic vector with one or more physiologically acceptable carriers or excipients. For example, the therapeutic vector may be ded in a carrier such as PBS (phosphate buffered saline). The active compounds can be administered by any appropriate route, for example, orally, parenterally, intravenously, ermally, subcutaneously, or topically, in liquid, semi-liquid or solid form and are formulated in a manner suitable for each route of administration.

In some embodiments, the therapeutic vector and physiologically acceptable salts and solvates are formulated for administration by inhalation or insufﬂation (either through the mouth or the nose) or for oral, , parenteral or rectal administration. In some embodiments, for administration by inhalation, the therapeutic vector is delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodiﬂuoromethane, trichloroﬂuoromethane, dichlorotetra-ﬂuoroethane, carbon dioxide or other suitable gas. In some embodiments, a pressurized aerosol dosage unit or a valve to deliver a metered . In some embodiments, capsules and cartridges (e.g., of gelatin) for use in an r or insufﬂator are formulated containing a powder mix of a therapeutic compound and a le powder base such as lactose or starch.

In some embodiments, the pharmaceutical itions are ated for oral administration as tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as g agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g, magnesium stearate, talc or ); disintegrants (e.g, potato starch or sodium starch ate); or wetting agents (e.g., sodium lauryl sulphate). In some embodiments, the tablets are coated by methods well known in the art.

In some embodiments, liquid preparations for oral administration are in the form of, for example, solutions, syrups or suspensions, or they are formulated as a dry product for constitution with water or other suitable vehicle before use. In some embodiments, such liquid preparations are prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily , ethyl alcohol or fractionated vegetable oils); and vatives (e.g., methyl or propyl-p- hydroxybenzoates or sorbic acid). In some embodiments, the preparations also contain buffer salts, ﬂavoring, ng and sweetening agents as appropriate. In some embodiments, pharmaceutical compositions are formulated oral administration to give controlled release of the active compound. In some embodiments, the pharmaceutical compositions are ated for buccal in the form of tablets or lozenges formulated in conventional manner.

In some embodiments, the therapeutic vector is formulated for parenteral administration by injection, e.g., by bolus ion, or continuous infusion. In some embodiments, formulations for injection are in unit dosage form, e.g., in ampoules or in multi- dose ners, with an added preservative. In some embodiments, the compositions are formulated as suspensions, solutions or emulsions in oily or aqueous vehicles. In some embodiments, the ations comprise formulatory agents such as ding, stabilizing and/or dispersing . Alternatively, in some embodiments, the active ingredient is in powder lyophilized form for constitution with a suitable vehicle, e. g., sterile pyrogen-free water, before use.

In some embodiments, the therapeutic vector is formulated as a depot preparation. In some embodiments, such long acting ations are administered by tation (for example, subcutaneously or intramuscularly) or by uscular injection.

Thus, for example, in some embodiments, the therapeutic compounds are formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly e derivatives, for example, as a gly soluble salt.

In some ments, the active agents are formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracistemal or pinal application. In some embodiments, such solutions, particularly those intended for ophthalmic use, are formulated as 0.0l%-10% isotonic solutions, pH about 5-9, with appropriate salts. In some embodiments, the compounds are formulated as aerosols for topical application, such as by inhalation.

] The concentration of active compound in the drug composition will depend on absorption, inactivation and excretion rates of the active compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.

In some embodiments, the compositions are presented in a pack or dispenser device which comprise one or more unit dosage forms containing the active ingredient. In some embodiments, the pack may comprises metal or plastic foil, such as a blister pack. In some embodiments, the pack or dispenser device is accompanied by instructions for administration.

In some embodiments, the active agents are packaged as es of manufacture containing packaging material, an agent provided herein, and a label that tes the disorder for which the agent is provided.

Animal Models In some embodiments, the retroviral vector particles, hereinabove described are administered to an animal in vivo as part of an animal model for the study of the effectiveness of a gene therapy treatment. In some embodiments, the retroviral vector particles are administered in varying doses to different animals of the same species. The s then are evaluated for in viva expression of the desired therapeutic or diagnostic agent. In some embodiments, from the data obtained from such evaluations, a person of ordinary skill in the art determines the amount of retroviral vector particles to be stered to a human patient.

Also provided are kits or drug delivery systems comprising the compositions for use in the methods described herein. All the essential materials and reagents required for stration of the retroviral particles disclosed herein may be assembled in a kit (6.g. packaging cell uct or cell line, cytokine sion vector). The components of the kit may be provided in a variety of formulations as bed above. The one or more therapeutic retroviral particles may be ated with one or more agents (e.g., a chemotherapeutic agent) into a single pharmaceutically acceptable composition or separate pharmaceutically acceptable compositions.

The components of these kits or drug delivery systems may also be provided in dried or lyophilized forms. When reagents or components are provided as a dried form, reconstitution generally is by the addition of a suitable solvent, which may also be provided in another container means.

Container means of the kits may generally include at least one vial, test tube, ﬂask, bottle, syringe and/or other container means, into which the at least one substance can be placed.

The kits disclosed herein may also comprise ctions regarding the dosage and/or administration information for the retroviral particle. Instructions can include ctions for practicing any of the methods described herein including treatment methods. Instructions can additionally include indications of a actory clinical endpoint or any e symptoms that may occur, or additional information required by regulatory agencies such as the Food and Drug stration for use on a human subject.

WO 53258 2014/029814 The instructions may be on “printed matter,” e.g., on paper or cardboard within or afﬁxed to the kit, or on a label afﬁxed to the kit or packaging material, or attached to a vial or tube containing a component of the kit. Instructions may additionally be included on a er readable medium, such as a disk (ﬂoppy diskette or hard disk), optical CD such as CD- or DVD- ROM/RAM, magnetic tape, electrical e media such as RAM and ROM, IC tip and hybrids of these such as magnetic/optical e media.

In some ments, the kits or drug delivery systems include a means for ning the vials in close ment for commercial sale such as, e.g., injection or blow- molded plastic containers into which the desired vials are retained. Irrespective of the number or type of containers, the kits may also se, or be packaged with, an instrument for assisting with the inj /administration or placement of the ultimate complex composition within the body of a subject. Such an instrument may be an applicator, inhalant, e, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle.

Packages and kits can further include a label specifying, for example, a product description, mode of administration and/or indication of ent. Packages provided herein can include any of the compositions as described herein. The package can further include a label for treating one or more diseases and/or conditions.

The term “packaging material” refers to a physical structure housing the components of the kit. The packaging material can maintain the components sterilely and can be made of material commonly used for such purposes (e.g., paper, corrugated ﬁber, glass, plastic, foil, ampules, etc). The label or packaging insert can include appropriate written instructions.

Kits, therefore, can additionally include labels or instructions for using the kit components in any method described herein. A kit can e a compound in a pack, or dispenser together with instructions for administering the compound in a method described herein.

EXAMPLES In order that those in the art may be better able to practice the compositions and methods described herein, the following examples are provided for illustration purposes.

Example 1: Cell Line Generation First, iral supernatant is generated by transfection of a 3 or 4 d system with calcium phosphate t into 293 T cells. Supernatant is ﬁltered through a 0.45 um ﬁlter. Filtered supernatant can be used fresh, stored up to 48 hours at 40 C, or stored at -80° Cell lines are generated by seeding l x 104 cells/well in a 6 well tissue culture dish. The next day retroviral supernatant is added with 8 ug/mL polybrene for 16-24 hours and WO 53258 selected with the appropriate dose of selection drug (G418 ,hygromycin or puromycin). The dose of the selection drug is the minimum amount to cause 100% kill on V-TK cells at least 4 days post on of drug, in order to avoid excessive toxicity to cells.

Example 2: GCV Sensitivity Assay Cells expressing HSV-TK, or a mutant and/or variant thereof, are seeded at l X 105 in 6 well dishes. The next day, 5 serial 10 fold dilution of GCV are added with a ﬁnal concentration ranging from 1 mM to 01 um. Three (3) days after GCV treatment, methylene blue is added to stain live cells.

Example 3: Bystander Assay Cells are seeded at l-4 x 104 cells/well in a 96 well plate, in triplicate, with mixtures of TK cells ranging from 0-100%. The next day GCV is added at doses ranging from um to 1 mM. Cells plates at conﬂuency are split 1:30 into 3 , 20-24 hours after GCV on. 5 days later, cells are analyzed by Presto Blue for live cell metabolism and read on a microplate reader. Cell plates at sub-conﬂuency are analyzed 3 days after GCV treatment by Presto Blue.

In one assay, the inventors used HSV-TK clonal cell lines were generated; using Neomycin-HSV-TK, Hygromycin-HSV-TK, Red Fluorescent protein (RFP)-HSV-TK cell lines and several mutants of HSV-TK gene were compared.

] RexC2 carries an improved version of the Herpes simplex virus (HSV) Thymidine Kinase gene (TK). A cellular host that has been efﬁciently infected duced) with RxC2 will integrate the viral TK in its genome and express this enzyme. HSV-TK phosphorylates the DNA base thymidine for its incorporation into newly synthesized DNA in dividing cells.

A typical 96-well plate plan for cell g and GCV treatment is shown in the table below (HK=HSV-TK): NO NO NO NO media 20 uM 20 uM 20 uM 10 uM 10 uM 10 uM GCV GCV GCV GCV III-“mu nmedia media media media media media media media media media media 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% media TK TK TK TK TK TK TK TK TK TK IIIIIIIIIIIICmedia TK TK TK TK TK TK TK TK TK TK uIIIIIIIIIIImedia TK TK TK TK TK TK TK TK TK TK nIIIIIIIIIIImedia TK TK TK TK TK TK TK TK TK TK % 25% 25% 25% 25% 25% 25% 25% 25% 25% I: media 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% media TK TK TK TK TK TK TK TK TK TK nmedia media media media media media media media media media media Graphic results of a bystander assay experiment are shown in Figures 19 and 20.

] More than 40 bystander assays were performed using different mutants HSV-TK and clonal populations.

The data was compiled with GCV sensitivity and enzyme kinetics measurements and viral titer of tion for the mutants with potential.

The careful examination of all these parameters allowed the selection of the mutant HSV-TK168dmNES to be the TK gene in Reximmune C-2.

Exam le 4: uantitation of S liced Form of TK RNA b Real Time PCR The unspliced and truncated form of HSV-TK are subcloned into a pCR2.l TOPO vector (Invitrogen). Two quantitative real time PCRs are set-up with two different sets of primers and probes able to selectively amplify and detect the unspliced and spliced form of HSV-TK, using the TaqMan®/ABI PRISM 7700 sequence detection system. For the HSV-TK unspliced form, primers and probe are designed in the spliced region of the HSV-tk gene Real Time PCR for the unspliced form is med in a 25 ul reaction e containing 100-500 ng of genomic DNA or 10 ul of cDNA, 1X TaqMan® Universal PCR Master Mix, 300 nM of each of the two primers TKwtfor (5'-CGG CGG TGG TAA TGA CAA G-3') and Tkwtrev (5'-GCG TCG GTC ACG GCA TA-3') and 200 nM of TKwt MGB probe (5'- FAM CCA GAT AAC AAT GGG C-3').

A ® probe assing the splice on is designed to selectively detect the HSV-TK spliced form. Quantitative Real time PCR speciﬁc for the TK spliced (truncated) form was performed in a 25 ul reaction mixture containing 100-500 ng of genomic DNA or 10 ul of cDNA, 1X Master Mix (PE Applied Biosystems) 300 nM of each of the two primers. l cycling conditions are as follows: l activation ofUNG at 50 0C for 2 min, followed by activation of Taq Gold and inactivation ofUNG at 95 0C for 15 min. Subsequently, 40 cycles of ampliﬁcation are performed at 95 0C for 15 s and 60 0C for l min. Both PCRs are performed in parallel in MicroAmp® optical 96-well reaction plates (Applied Biosystems) using the ABI Prism 7700 ce Detection Systems (Applied Biosystems). Mean baseline ﬂuorescence was calculated from PCR cycles 3 to 15, and Ct was deﬁned as the PCR cycle in which the normalized ﬂuorescence intensity of the er dye d 0.05. Two standard curves with known copy numbers (from 10<6 >to 4 copies/reaction) are generated in each TaqMan® assay by plotting the Ct values against the logarithm of the initial input ofDNA amount. Standard dilutions and cDNA samples are analyzed in duplicate and triplicate, respectively.

Example 5: Clinical Trial A dose escalation trial was conducted to evaluate the safety, pharmacokinetics, and pharmacodynamics of Reximmune-C2 dine Kinase and GM-CSF Genes) in refractory subjects with primary hepatocellular carcinoma or tumors metastatic to the liver.

Background and Rationale Reximmune-C2 is sed of a genetic delivery platform containing an internal d that encodes for eutic proteins of interest. The c delivery platform has been dosed in over 280 subjects worldwide; approximately 270 subjects were treated with the vector containing dnGl as a payload -G) and 16 subjects with thymidine kinase (vTK) and the immune ator Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) as a payload (Reximmune-C). The genetic ry platform is a highly engineered combinant Mouse Moloney Viral vector (MoMLV). Previously, a Phase 1 dose tion trial was performed investigating the combination of Rexin-G and Reximmune-C in subjects with refractory primary or metastatic solid tumors (Genevieve Trial). This proposed Phase I al trial (entitled eve 2 Trial) is an extension of a trial undertaken investigating Reximmune- C2 alone — without the Rexin-G — utilizing an improved form of thymidine kinase in a thymidine kinase plus GM-CSF combination.

In the original Genevieve trial, sixteen subject were recruited over 3 dose levels with the mean exposure in the t dose group being 8.0x 1010 cfus (# of pts = 7) and the longest duration 6 cycles (range of cycles 3-6). For Part A of the study, treatment consisted of a previously ined safe and effective (optimal) dose of Rexin-G, and escalating doses of Reximmune-C. Specifically, Rexin-G, 2 x 1011 cfu, on Days 1, 3, 5, 8, 10 and 12, Reximmune- C, 1.0, 2.0 or 3.0 x 1010 Cﬁl on Day 3 (Dose Levels 1, II, 111 respectively), and valacyclovir at 1 gm p.o. three times a day on Days 6-19, as one cycle. For the Part B part of the study, subjects who had no toxicity or in whom toxicity had resolved to Grade 1 or less could receive additional cycles of therapy up to a total of 6 treatment cycles.

There were no dose-limiting toxicities at any dose level. Unrelated adverse events were reported for the 16 subjects in the study, but the number of events was low (in most cases 1 or 2 occurrences per preferred term), and most were Grade 1 or 2. Related non-serious e events occurred in 2 subjects and both were Grade 2. Four subjects experienced serious adverse events, all of which were deemed not related to the study drug.

The rationale for uation of this Phase 1 trial is that: (l) thymidine kinase itself could prove to be an effective anticancer agent particularly in subjects whose tumors demonstrate a bystander effect; (2) stration of the genetic delivery platform to date to an international group of subjects has demonstrated a very high degree of safety; and (3) biodistribution in animals suggests a high biodistribution to the liver. Moreover, the addition of GM-CSF could bute to an immunological effect and enhanced tumor cell kill through tumor associated antigens through recruitment of the appropriate immune cells.

The tribution of the viral particles is highest to the liver, followed by , then lung — this is the rationale for focusing initially on hepatocellular tumors where the dose intensity should be the highest. There is also a high clinical unmet need for effective anticancer agents for these cancers.

It is understood that the embodiments disclosed herein are not limited to the particular methods and components and other processes described as these may vary. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include the plural reference unless the context clearly dictates ise. Thus, for example, a nce to a "protein" is a reference to one or more ns, and includes equivalents f known to those skilled in the art and so forth.

Unless deﬁned ise, all technical and scientiﬁc terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Speciﬁc methods, s, and materials are described, although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the t invention.

All publications cited herein are hereby incorporated by reference including all journal articles, books, manuals, published patent applications, and issued patents. In addition, the meaning of certain terms and phrases employed in the speciﬁcation, examples, and appended claims are provided. The deﬁnitions are not meant to be limiting in nature and serve to provide a clearer understanding of certain aspects of the t invention.

Example 6: Clinical Trial for gene y applications.

This clinical trial is divided into two phases: Phase IA in which Reximmune-C2 was administered as a single intravenous dose on three out of ﬁve days . Valganciclovir (the oral form of lovir) dosing is ted on day 8 for 5 days irrespective of the PET scan results.

An approximately one week drug holiday follows. Each cycle will be of three weeks duration.

There will be three patients in the ﬁrst and subsequent s until a t experiences Dose Limiting ty (DLT) or two instances ofNCI-CTC Grade 2 ties attributed to the study drug (except nausea/vomiting, fatigue, anorexia, alopecia, or anemia). If there are no DLTs, patients will move to the next dose level. If there is a DLT, the cohort will be expanded to 6 ts and the dose level will not be exceeded if 2 or more patients exhibit DLTs.

Once the Maximum Administered Dose (MAD) is reached, a d Fibonacci schedule will be followed starting with the cohort dose which had no DLTs and continuing until dose-limiting toxicities are observed in two ts at a dose level. Once the Recommended Phase 2 Dose (RP2D) is deﬁned, 6-12 ts will be recruited.

Phase IB is designed to explore the activity of Reximmune-C2 in ts of a deﬁned tumor type and stage based on the Phase IA data and who are [18F]FHBG scan ve day three to six after one dose (RP2D) of Reximmune-C2. If the scan is positive, the t is accepted into the Phase IB treatment phase of the protocol and the RP2D is given as three doses within 5 days, followed by 5 days of valganciclovir beginning on day 8 of that phase, followed by a one week drug holiday. Each cycle is of three week duration. Patients who have a negative [18F]FHBG PET scan after one single dose of Reximmune-C2 will be dosed with 5 days of valganciclovir and will not continue in the study.

The patient DLT will be deﬁned as the occurrence of any of the following events which is attributed to Reximmune-C2 and occurring during the ﬁrst cycle (3 weeks) of drug administration: Grade 4 neutropenia (i.e., absolute neutrophil count (ANC) < 500 cells/mm3) for 7 or more consecutive days or febrile neutropenia (i.e., fever 2 38.50 C with an ANC < 1000 cells/mm3); Grade 4 thrombocytopenia (< 25,000 cells/mm3 or bleeding episode requiring platelet transfusion); Grade 3 or greater nausea and/or vomiting despite the use of adequate/maximal medical intervention and/or prophylaxis; Any Grade 3 or greater nonhematological toxicity (except Grade 3 ion site reaction, alopecia, fatigue); Retreatment delay of more than 3 weeks due to delayed recovery from a toxicity related to treatment with Reximmune-C2; and Grade 3 or greater hypersensitivity reaction despite the appropriate use of premedications (by Common Toxicity Criteria deﬁned as omatic bronchospasm, requiring parenteral medications(s), with or without urticaria; allergy-related edema- angioedema”).

Reximmune-C2 is infused intravenously over 15-60 minutes (depending on the dose) Via an infusion pump. une-C2 is provided in 30 ml vials stored at -80 °Ci 10 0C.

WO 53258 In this Phase I trial, the safety, pharmacokinetics, and pharmacodynamics of escalating doses of Reximmune-C2 will be investigated. The maximum tolerated dose will be identiﬁed and a recommended Phase 2 dose will be deﬁned for Reximmune C2. Any antitumor activity and clinical responses to Reximmune-C2 treatment will be described.

The starting dose in this trial is based on: human clinical safety experience with the related vector platform drug products Rexin-G and Reximmune-C and the results of the 21 day rat GLP toxicology study for Reximmune-C2.

Objectives The primary objective of the study is to determine the m tolerated dose (MTD), dose limiting toxicity (DLT), safety, and a ended Phase 2 dose (RP2D) of Reximmune-C2 administered over a three week cycle consisting of a series of three doses given intraveneously within ﬁve days in week 1, followed by 5 daily doses of valganciclovir in week 2 in patients enrolled in this study who have been diagnosed with advanced primary or metastatic tumors to the liver.

Secondary objectives include: (i) evaluation of the plasma pharmacokinetics of une-C2; (ii) assessment of the surrogate of HSV-TK-m2 n expression from Reximmune-C2 via serial [18F]FHBG PET and/or SPECT g; (iii) description and assessment of any preliminary evidence of anti-tumor activity of Reximmune-C2; and (iv) to provide clinical research testing for dies to ector gp70 env, replication-competent retrovirus in peripheral blood cytes (PBLs); vector integration into genomic DNA of PBLs, and circulating hGM-CSF protein.

Methods Study Design: Parallel group, open label dose escalation, three-center clinical trial.

Stratiﬁcation: None.

Therapy: Reximmune-C2 will be administered as an intravenous infusion to separate patients. In Phase IA — investigating Reximmune-C2 - the dose will be escalated among cohorts of patients until DLT is observed. At the RP2D, additional patients will be recruited. In Phase IB ts will be pre-screened by [18F]FHBG PET for expression of the HSV-TK-m2. Those that express HSV-TK-m2 will receive additional doses of Reximmune-C2.

Patients will not be pre-medicated unless hypersensitivity ons occur.

Statistical Methods: ptive statistics will be used for tical analysis.

Sample Size Determination: Precise sample size cannot be deﬁned, as it is dependent on the observed toxicity. For each schedule, cohorts of three to six subjects will be treated at each dose level until the MTD is deﬁned. Once the MTD is identiﬁed, this dose level will be expanded to a maximum of 12 patients who will be treated to better deﬁne the tolerability and pharmacokinetics of the dose and schedule. It is expected that 45-70 subjects will be enrolled, with 33 to 46 in the IA portion.

Enrollment ia Subjects must meet all of the following inclusion criteria to be eligible for randomization into the study: 1. Diagnosis of histologically documented, advanced stage, primary or metastatic adult solid tumors in the liver that are refractory to standard therapy or for which no curative standard therapy exists. 2. Evidence of radiographically measurable or evaluable disease. 3. All acute toxic effects of any prior radiotherapy, chemotherapy, or surgical procedures must have resolved to National Cancer Institute (NCI) Common Toxicity Criteria (CTC)(Version 4.0) Grade < 1. 4. Age must be > 18 years. 5. Last dose of antineoplastic therapy except for hormonal therapy must be > 21 days. External beam radiotherapy must have been < 25% bone marrow-containing on. 6. Patients may be tis B and C positive. (Patients may continue their antiviral medications). ] 7. Patients may have intracranial metastases of any number if they have been brain irradiated and stable for 6 weeks. Patients may be taking anti-seizure medicines but must not be on steroids. 8. ky performance status must be 2 70. 9. Life ancy of at least 3 months. 10. Patients must be able to travel to St. Luke’s Medical Center for the PET scans. ll. Required ne laboratory data include: Absolute neutrophil count 3 _ 9 2 mm [SI units 10 /L] (ANC) obin 2 8.0 gm/dL [SI units mmol/L] Serum Creatinine S 1.5 x laboratory upper limit of normal (L-ULN) Bilirubin S 2.0 mg/dL Alkaline phosphatase S 5 x L-ULN AST, ALT S 5 x L-ULN LDH S 5 x L-ULN Pregnancy test (females of Negative within 7 days of starting Protocol childbearing potential) 12. Signed informed consent indicating that they are aware of the neoplastic nature of their disease and have been informed of the procedures to be followed, the experimental nature of the therapy, alternatives, potential beneﬁts, side effects, risks, and discomforts. 13. Willing and able to comply with scheduled visits, treatment plan, and laboratory tests.

The ce of any of the following will exclude a subject from study enrollment ] 1. Concurrent therapy with any anticancer therapy including any other igational agent. 2. Known intracranial edema or a CVA within 6 weeks of screening. 3. Pregnant or breast-feeding women. Female subjects must agree to use ive contraception, must be surgically sterile, or must be postmenopausal. Male subjects must agree to use effective contraception or be surgically sterile. The deﬁnition of effective contraception will be based on the nt of the Investigator or a designated associate. All at- risk female subjects must have a negative ncy test within 7 days prior to the start of study treatment. 4. Clinically significant cardiac e (New York Heart Association, Class III or IV). 5. ia or altered mental status that would prohibit informed consent. 6. Other severe, acute, or chronic l or psychiatric condition or laboratory ality that may increase the risk associated with study participation or study drug stration or may interfere with the interpretation of study results and, in the judgment of the pal Investigator, would make the subject inappropriate for this study. 7. Known side effects to antivirals in the ganciclovir class. 8. Patients who are known to be HIV positive. 9. Patient must not be taking steroids at the time of screening.

Rationalefor the Starting Dose and Schedule Reximmune-C has been dosed in 16 patients over a range of 1.0, 2.0 or 3.0 x1010 cfu (Dose Levels 1, II, 111 respectively on day 3 of the cycle). There were no dose-limiting toxicities at any dose level. Unrelated adverse events were reported for the 16 patients in the study, but the number of events was low (in most cases 1 or 2 occurrences per preferred term), and most were Grade 1 or 2. Related nonserious adverse events occurred in 2 patients and both were Grade 2. Four patients experienced serious adverse events, all of which were deemed not related to the study drug. The trial was closed prior to determining the optimal dose and schedule of Reximmune-C. In this trial, the new Genevieve-2 Trial, initial dosing will be based on the 21 day toxicology and the HSV-TK-ml study. Future dosing will proceed using total viral particles (TVP)/ml which is a more accurate measure of titer than cfu per mL.

The le is based on the rationale that Reximmune-C2 re will not uce all of the tumor cells. Therefore, patients will be dosed three times in a cycle over a period of 5 days.

The time n exposure to GDS and the expression of HSV-TK-m2 (and hGM-CSF) is estimated to be 48 to 72 hours. Therefore, 72 hours after the third dose of Reximmune-C2, valganciclovir will be initiated. The dose (which will be adjusted for renal on) will be given at conventional antiviral dose levels. Due to the ial toxicity of valganciclovir and the published observations that 5 days of lovir should be sufﬁcient to kill the majority of cells containing HSV-TK-m2, 5 days of therapy was chosen. Due to the ial toxicity of both Reximmune-C2 and valganciclovir, this will be followed by an approximately 9 day drug holiday. The hGM-CSF may be at sufficient concentrations at the time of valganciclovir addition to inﬂuence the presentation of any tumor ated antigens (TAAs) that may appear during tumor cell sis.

Plasma samples will be taken after the first and third doses in Cycle One and after the ﬁrst dose in Cycle Two for pharmacokinetics.

As distribution is primarily to the liver, toxicities will be carefully monitored there and because of the implications, the bone marrow.

This clinical protocol calls for the administration of Reximmune-C2 via intravenous on to patients with advanced malignancies, either primary hepatocellular or tumors metastatic to the liver. There will be two parts: Phase IA (dose escalation 3 doses/week every three weeks) and Phase IB (pre-screening after one dose of Reximmune-C2 and an [18F]FHBG scan). If the PET scan is positive, the patient will ue on study. If the PET scan is negative, the patient will receive 5 days of valganciclovir and will not continue in the trial. For Phase IA, dose escalation will follow an accelerated titration design, incorporating three ts per dose level until either one instance of DLT or two instances ofNCI-CTC Grade 2 toxicities attributed to the study drug t nausea/vomiting, fatigue, anorexia, alopecia or anemia) are observed. Thereafter, dosing in the clinical protocol will follow a d Fibonacci schedule until dose-limiting toxicities are achieved.

Trial Design This is a Phase 1, abel, four center, scalating trial. The dose will be increased until DLT is observed, and the MTD is deﬁned.

Reximmune-C2 will be administered as an IV inﬁlsion over 15-60 minutes. It is pated that 33-70 patients will be d during the course of the study.

For Phase IA, the dose of Reximmune-C2 will be escalated from 11 TVP.

In the accelerated dose escalation phase, cohorts of three patients will be enrolled at each dose level. The dose tion increment will be 100% until a DLT or two CTC Grade 2 or greater toxicities are observed. When the accelerated dose escalation ends, the dose escalation for a new patient in the standard dose escalation will follow a modiﬁed Fibonacci scheme (i.e., dose increments of 67%, 50%, 40%, 33% and 25%). A minimum of three patients per dose level will be enrolled. For Phase 1B, the dose of une-C2 will be the RP2D. DLT will be assessed.

If a DLT is observed in 3 2 out of six patients at a dose level, there will be no ﬁarther dose escalation; this dose level will deﬁne the maximum administered dose (MAD).

The dose just below the MAD will be considered the MTD. Once the MTD is deﬁned, this dose level can be expanded to a maximum of twelve patients to ﬁarther characterize the cokinetic and pharmacodynamic parameters and suitability as a recommended dose for Phase 2 clinical studies.

Treatment of Patients Only qualiﬁed personnel who are familiar with procedures that minimize undue exposure to themselves and to the environment should undertake the preparation, ng, and safe disposal of biotherapeutic agents in an appropriate environment.

Reximmune C2 is a Moloney Murine replication incompetent retrovector particle containing the genes encoding for a HSV-TK-m2 and hGM-CSF. The drug product contains DMEM (low glucose), RD-Retrovector Particles, L-glutamine, Sodium pyruvate, human serum albumin, n-butyric acid, Pulmozyme®, magnesium and other ents.

Drug product is available in one vial size: 30 mL type 1 clear glass vials with a mm ﬁnish (containing 25 mL of 31 0 TVP). The vials are closed with 20 mm Teﬂon coated serum stoppers and 20 mm ﬂip-off lacquered ﬂip tops.

Reximmune-C2 will be administered intravenously by lI‘lﬁISlOIl pump over 15 minutes up to a volume of 100 mL, from >100 mL to 200mL over 30 minutes, from >200 mL to 300 mL over 45 minutes, and from >300 mL to 400 mL over 60 minutes. s over 400 mL will be administered at a rate determined by the Investigator and the Gleneagles Medical Monitor. Once the MTD has been identiﬁed for the schedule, the time of administration may be changed, if indicated (and as agreed between the Investigator and the Gleneagles Medical Monitor).

Valganciclovir is administered orally, and should be taken with food. Serum creatinine or creatinine clearance levels should be red careﬁJlly. Dosage adjustment is required based on nine nce as shown in the Table below. Valganciclovir dosing may begin on day 7 to 9 of the cycle but must be given for 5 consecutive days.

Creatinine clearance can be calculated from serum creatinine by the ing formula: ] For males = {(140 — age[years]) X (body weight [kg])}/ {(72) X (0.011 X serum nine [micromol/L])} For females = 0.85 X male value.

Table I. Valganciclovir Dosing for Renally ed Patients Cr CL (ml/min) Dose Day 1 Dose Days 2 - 5 260 ml/min 900 mg (two 450 mg tablets) 900 mg (two 450 mg bid tablets) qday 40-59 ml/min 450mg bid 450mg qday -39 ml/min 450mg 450 mg Day 3 and Day 5 -24 ml/min 450mg 450 mg Day 4 The e of the Phase 1 study is to establish the MTD, DLT, safety and a RP2D of the investigational agent. T0Xic effects are thus the primary study endpoint and will be assessed continuously. Response information will be obtained if patients have disease that can readily be measured and re-assessed. These assessments will be made with every cycle. rmore, a response must be noted between two examinations at least 6 weeks apart in order to be documented as a conﬁrmed response to therapy. 0 Evaluable for toxicity - All patients will be evaluable for toxicity if they receive any study drug. 0 Evaluable for response - All patients who have received at least a single cycle of treatment and had tumor re-assessment will be considered ble for response. In addition, those patients who develop early progressive disease will also be considered evaluable for response. Patients on therapy for at least two cycles of treatment will have their response evaluated.

The determination of antitumor efﬁcacy will be based on ive tumor assessments made according to the Immune-Related Response Criteria (irRC) system of evaluation and treatment decisions by the Investigator will be based on these assessments.

Given the presence of the GM-CSF transgene in Reximmune-C2 and the possibility of an immune response contributing to the tumor effect, the Immune response Criteria will be utilized for clinical response. The reasons for using The immune se Criteria vs RECIST 1.1 are as follows: (1) the ance of measurable umor ty may take longer for immune therapies than for xic therapies; (2) responses to immune therapy occur after conventional PD; (3) discontinuation of immune therapy may not be appropriate in some cases, unless PD is conﬁrmed (as is usually done for response); (4) allowance for “clinically cient” PD (e. g. small new s in the presence of other responsive lesions) is recommended; and (5) durable SD may represent antitumor activity.

The comparisons between RECIST 1.1 and the Immune-Related Response Criteria are listed below: Table 11. Comparison ofWHO RECIST and Immune-Related Response Criteria WHO irRC New measurable lesions Always represent PD Incorporated into tumor burden (i.e., 2 5 X 5 mm) New, nonmeasurable lesions Always represent PD Do not deﬁne progression (but (i.e., < 5 X 5 mm) preclude irCR) Non-index lesions Changes bute to deﬁning Contribute to deﬁning irCR BOR of CR, PR, SD, and PD (complete earance required) CR Disappearance of all lesions in Disappearance of all lesions in two consecutive observations not two consecutive observations not less than 4 wk apart less than 4 wk apart PR 2 50% se in SPD of all 2 50% decrease in tumor burden index lesions compared with compared with baseline in two baseline in two observations at observations at least 4 wk apart least 4 wk apart, in absence of new lesions or unequivocal progression of non-index lesions SD 50% decrease in SPD compared 50% decrease in tumor burden with baseline cannot be compared with baseline cannot be established nor 25% increase established nor 25% increase compared with nadir, in absence compared with nadir ofnew lesions or unequivocal progression of non-index lesions PD At least 25% se in SPD At least 25% increase in tumor compared with nadir and/or burden compared with nadir (at unequivocal progression of non- any single time point) in two index s and/or appearance consecutive observations at least ofnew lesions (any any single 4 wk apart time point) Timing and Type of Assessments All baseline imaging-based tumor ments are to be performed within 14 days prior to the start of treatment. For the purposes of this study, all patients’ tumor assessments should be re-evaluated starting 9 weeks after initiation of ent and every 6 weeks thereafter (e. g., Week 9, Week 15, Week 21, etc.) for both Phase IA and Phase IB. All patients with responding tumors (irCR or irPR) must have the response conﬁrmed no less than 6 weeks after the ﬁrst documentation of response. All patients with tumor ssion must have progression conﬁrmed no less than 6 weeks after the ﬁrst documentation of progression.

The same method of assessment and the same technique should be used to characterize each identiﬁed and reported lesion at baseline and during -up. Imaging- based evaluation is preferred to evaluation by clinical examination when both methods have been used to assess the antitumor effect of treatment. All ements should be recorded in metric notation.

CT and CT/PET are the methods for tumor assessments. Conventional CT should be performed with cuts of 10 mm or less in slice thickness contiguously. Spiral CT should be performed using a 5 mm contiguous reconstruction algorithm. This applies to the chest, abdomen, and pelvis.

Chest CT will used for assessment of pulmonary lesions.

Clinical s will only be considered measurable when they are superﬁcial (e. g., skin nodules, palpable lymph nodes). In the case of skin lesions, documentation by color photography including a ruler to estimate the size of the lesion is recommended. ] [18F]FHBG PET-CT scans will be obtained after the patient receives the ﬁrst three doses of Reximmune-C2 (cycle 1) in Phase IA and after the screening dose of une-C2 in Phase IB. In Phase IA additional [18F]FHBG PET-CT scans can be obtained in subsequent cycles at the discretion of the Investigator and with approval of the Medical Monitor.

] Ultrasound should not be used to measure tumor lesions that are clinically not easily accessible for objective se evaluation, e. g., visceral lesions. It is a possible alternative to clinical ements of cial palpable nodes, SC lesions, and thyroid nodules. Ultrasound might also be useful to conﬁrm the complete disappearance of superﬁcial lesions usually assessed by clinical examination.

] Endoscopy, laparoscopy, and radionuclide scan should not be used for response assessment.

All patients’ ﬁles and radiological images must be available for source veriﬁcation and may be submitted for extramural review for ﬁnal assessment of mor activity.

Measurability of Tumor s At baseline, tumor lesions will be rized by the Investigator as measurable or non-measurable by the criteria as bed below: 0 Measurable: Lesions that can be accurately measured in at least one dimension (longest diameter to be recorded) as 2 20 mm with conventional techniques or as 2 10 mm with spiral CT scan. Clinical lesions will only be considered measurable when they are superﬁcial (e. g., skin nodules, palpable lymph nodes). 0 asurable: All other lesions, including small lesions (longest diameter < 20 mm with conventional techniques or < 10 mm with spiral CT scan) and bone lesions, leptomeningeal disease, ascites, pleural or pericardial effusions, lymphangitis of the skin or lung, abdominal masses that are not conﬁrmed and followed by imaging ques, cystic lesions, usly irradiated lesions, and disease nted by indirect evidence only (e. g., by laboratory tests such as alkaline phosphatase).

NOTE: Cytology and histology: If measurable disease is restricted to a solitary lesion, its neoplastic nature should be conﬁrmed by cytology/histology.

Response to therapy may also be assessed by independent, central, radiologic ] Recording Tumor Measurements All measurable lesions up to a maximum of 10 lesions, representative of all involved , should be identiﬁed as target lesions and measured and recorded at baseline and at the stipulated intervals during treatment. Target lesions should be selected on the basis of their size (lesion with the longest diameters) and their suitability for accurate repetitive measurements (either by imaging techniques or clinically).

] The longest diameter will be recorded for each target lesion. The sum of the longest diameter for all target lesions will be calculated and recorded as the baseline. The sum of the longest diameters is to be used as reference to ﬁthher characterize the objective tumor response of the measurable dimension of the disease during treatment. All measurements should be recorded in metric notation in centimeters.

All other lesions (or sites of disease) should be identiﬁed as non-target lesions and should also be recorded at baseline. Measurements are not required and these lesions should be ed as “present” or “absent.” Deﬁnitions of Tumor Response WO 53258 Immune-Related Response Criteria criteria will be followed for assessment of tumor response.

Determination of Overall Response by Immune-Related Response Criteria Target Lesions for Solid Tumors 0 Complete response (irCR) is deﬁned as the disappearance of all lesions (whether measurable or not, and no new lesions); conﬁrmation by a repeat, consecutive assessment no less than 6 weeks from the date ﬁrst documented. 0 Partial se (irPR) is deﬁned as a > 50% decrease in tumor burden relative to baseline ed by a consecutive assessment at least 6 weeks after the ﬁrst documentation. 0 Progressive disease (irPD) is deﬁned as a > 25% increase in tumor burden relative to nadir um ed tumor burden) conﬁrmed by a repeat, consecutive assessment no less than 6 weeks ﬁom the date ﬁrst documented lesions recorded since the treatment started, or the appearance of one or more new lesions. 0 Stable Disease (irSD) is deﬁned as not meeting the criteria for irCR or irPR, in absence of irPD.

Non-Target Lesions for Solid Tumors The cytological conﬁrmation of the neoplastic origin of any efﬁlsion that appears or worsens during ent when the able tumor has met criteria for response or irSD is mandatory to differentiate between response or irSD and irPD.

Conﬁrmation of Tumor se To be assigned a status of irPR or irCR, changes in tumor measurements in patients with responding tumors must be conﬁrmed by repeat studies that should be performed 3 6 weeks after the criteria for response are ﬁrst met. In the case of irSD, follow-up measurements must have met the irSD criteria at least once after study entry at a minimum interval of 6 weeks.

When both target and non-target lesions are present, individual assessments will be recorded separately. The l ment of response will involve all parameters as depicted in Table 111.

The best overall response is the best response recorded from the start of the treatment until disease progression/recurrence g as a reference for tumor progression the smallest measurements recorded since the treatment started). The patient’s best response assignment will depend on the achievement of both measurement and conﬁrmation criteria.

Patients will be deﬁned as being not evaluable (NE) for response if there is no post-randomization gic assessment. These patients will be d as failures in the is of tumor response data. al Efﬁcacy Assessment: Performance Status.

Patients will be graded according to the Kamofsky performance status scale.

Tumor Marker Response ] Method of Assessment While not a ﬁllly validated measure of y in many malignancies, serial determinations of tumor markers may allow tion of an easily performed, inexpensive, quantitative, clinical tool as a potential additional means for following the course of the illness during therapy.

A tumor marker decrease or se will not be assessed as an objective measure of e. In particular, a rising tumor marker value will not be considered in the deﬁnition of tumor progression, but should prompt a repeat radiographic evaluation to document whether or not radiographic tumor progression has occurred.

Calculated Endpoint Deﬁnitions Survival is deﬁned as the time from date of ﬁrst study drug treatment to date of death. In the absence of conﬁrmation of death, survival time will be censored at the last date of follow-up.

Tumor response rate is deﬁned as the proportion of patients who have any evidence of objective irCR or irPR.

TTP is deﬁned as the time from treatment to ﬁrst conﬁrmed documentation of tumor progression or to death due to any cause. For patients who do not have objective evidence of tumor progression and who are either removed from study treatment or are given antitumor treatment other than the study treatment, TTP will be censored. A tumor marker increase g criteria for tumor marker progression does not constitute adequate objective evidence of tumor progression. However, such a tumor marker increase should prompt a repeat radiographic evaluation to nt whether or not objective tumor progression has ed.

TTF is deﬁned as the time from treatment to ﬁrst ed documentation of tumor progression, or to off-treatment date, or to death due to any cause, ver comes ﬁrst.

Patients who are still on treatment at the time of the analysis and patients who are removed from therapy by their physicians during an objective response and who, at the off-treatment date, have no evidence for objective tumor progression will not be considered to have experienced treatment failure, unless the awal is due to the occurrence of a medical event. For these patients, TTF will be censored at the off-study date. Censoring for TTF will also be performed in those ts who are given antitumor treatment, other than the study treatment, before the ﬁrst of objective tumor progression, off-study date, or death. A tumor marker increase meeting ia for tumor marker progression does not constitute adequate objective evidence of treatment failure. However, such a tumor marker increase should prompt a repeat radiographic 2014/029814 tion to document whether or not objective tumor progression (and thus treatment failure) has occurred.

Time to ﬁrst deﬁnitive performance status worsening is the time from treatment until the last time the performance status was no worse than at baseline or to death, due to any cause, in the absence of previous documentation of deﬁnitive conﬁmed performance status worsening. For patients who do not have deﬁnitive performance status worsening and who are either removed from study or are given antitumor treatment other than the study treatment, deﬁnitive performance status worsening will be censored.

Time to ﬁrst deﬁnitive weight loss is deﬁned as the time from treatment until the last time the percent weight se from baseline was < 5% or to death due to any cause in the absence of previous documentation of ive weight loss. For patients who do not have ive weight loss and who are either removed from study or are given antitumor treatment other than study treatment, deﬁnitive weight loss will be censored.

Additional evaluations of the data may include best objective response, conﬁrmed and unconﬁrmed objective response rate, duration of study treatment, time to ﬁrst occurrence of new lesions, time to tumor response, stable disease at 24 weeks, and rate of progression free survival at 24 weeks. Data may be evaluated by RECIST 1.1 criteria, if needed.

Treatment stration Assessment For both Phase IA and 1B: dose intensity is deﬁned as the total dose/cycle times the number of weeks between start of treatment and last treatment plus 13 days.

Percent relative dose intensity is deﬁned as the proportion of the actual dose intensity divided by the planned dose intensity for that same period of time.

Example 7: RxCZ-GCV Kill Assay Kill assays were ted as follows. The percentage of cell kill by GCV after treatment with RXC2 depends on the infectability (transducibility) of the cancer cells tested.

Cells for each cell line were plated in a 6 well dish. The following day, the cells were uced with ector containg the EGFP (Enhanced Green Fluorescent Protein Gene) diluted 1:5. After 48 hours cells were collected. The ﬂuorescent and non-ﬂuorescent cells were d using an automated ﬂuorescent cells counter to determine the percent uced. The efﬁciency of transduction was examined using a virus ng the gene for Green ﬂuorescent protein where ransduction ncy is shown in decreasing order.

—«EGFP + cells <%> BREAST Hs578T 66 --/- 5 BREAST HCC-38 59 +/- 2.3 SKIN A375 57.7 --/- 7.1 LUNG NCI-H460 25.9 +/— 1.7 LIVER SkHepl 21.4 --/— 4 The same viral preparation was used for all cell lines shown here (titer 2.72E+lO TVP) Example 8: is of Reximmune—C2 mediated GCV kill of cell lines expressing PiT-2 Cell lines expressing PiT-2 were established by transduction of target cells with a E-Rex expression retroviral vector containing the PiT-2 and Neomycin Resistance genes. Stable cell lines were then drug selected (G418) to establish a pure population of PiT-2 expressing cells. The cell lines were veriﬁed by amphotropic retrovial vector transduction of the LUC-2 gene into PiT2 expressing cells followed by bioluminescent analysis. For Reximmune-C2 cell kill is, PiT2 expressing cell lines were then plated in 48 well plates. The following day cells were transduced with the Reximmune-C2 retrovector. After transduction, cells were exposed to a daily dose of 20-40 uM GCV. After four days of GCV treatment the cells were analyzed for cell ity using the PrestoBlue reagent. This reagent is a resazurin-based solution that in the presence of the reducing nment of viable cells converts the t into ﬂuorescence that is quantitated using absorbance measurements.

Human colon cancer lines HCT-lS demonstrated poor HSV-TK-GCV kill and RKO cell line demonstrated no cell kill following Reximmune-C2 transduction and GCV exposure. PiT-2 expressing HCT-lS and RKO lines were ted and their transduction efﬁciency examined; resutls are ed in the following table.

Cell Line EGFP+ cells (%) PiT—2-CHO-K1 34 +/- 2.9 PiT—2-MIA-PaCa-2 78.6 +/- 2.2 PiT—2-HA-HCT-15 14.9 +/- 1.2 PiT—2-RKO 43.1 +/- 1.6 A considerable increase of LNCE-RVE transduction efficiency was observed in all PiT-2 expressing cell lines demonstrating that the kill activity is increased when target cells express PiT2.

Using cells lines expressing PiT-2, the data shows that the requirement of PiT-2 receptor presence for LNCE-RVE transduction is reﬂected by the level of EGFP expression in cells ed by cent microscopy (data not shown). The requirement of PiT-2 presence for Reximmune-C2 infectivity has also been shown by a GCV cell kill assay. Therefore, PiT-2 represents a good biomarker for Reximmune-C2.

It was determined that Pit2 expression correlated to Reximmune-C2 mediated GCV cell kill. HSV-TK-GCV kill of CHO-Kl parent line versus PIT2 expressing CHO-Kl lines.

Figures 25 and 26 e graph results -GCV kill after single or triple transduction in various cell lines following single or triple transduction in the absence of PiT-2 (panel A of each figure) or presence of PiT-2 (panel B of each figure).

] Figure 27 es the results of TK-GCV kill after triple transduction with Reximmune-C2 in a MIA-PaCa-2 human atic carconima cell line. GCV kill was ive at the higher concentrations of TVP.

Figure 28 provides the results of HSV-TK-GCV kill after triple transduction of PiTMIA-PaCa-2 cells with Reximmune-C2. GCV kill of RxC2-triple transduced PiTMIA- PaCa2 human atic carconima cell line. The presence of PiT-2 dramatically increased the amount of cell killing at lower concentrations of TVP.

Figure 32 illustrates a graph of RxC2-tranduced CHO-Kl cell lines after four days in GCV.

Figure 33 rates a graph of RxC2-tranduced PiTHA-CHO-Kl cell lines after four days in GCV.

It is very apparent that, even at the lowest concentration of GCV, the presence of PiT-2 allows for significantly greater transduction and cell killing.

Example 9: Transduction ncy versus GCV Kill After Reximmune C2 triple transduction To demonstrate transduction efficiency and GCV kill, cells were plated into 48 well plates. The next day cells are transduced with Reximmune-C2 diluted in the range of l :40 to 1:5120. Following the last of three transductions, the cells were exposed a daily doses of GCV (20-40 uM) for four days. One day following the last dose of GCV the cells were analyzed using the Prestoblue reagent for cell viability. This reagent is a resazurin-based on that in the presence of the reducing environment of viable cells converts the reagent into ﬂuorescence that is quantitated using absorbance measurements. The results are ed as percent kill based on the non-transduced cell viability.

A549 16.3 --/- 1.5 Figure 31 is a graph depicting the percentage of GCV kill after Reximmune-C2 triple transduction of various cancer cell lines. The graph demonstrates the variation in GCV kill amongst the different cell lines. The cell lines are comparable across each dilution converted to the total virus les/mL against the percent cell kill. The table gives the transduction efﬁciencies for the cell lines ented in the graph. The percent ncy does not seem to have a direct correlation with the cell kill, but a trend is evident in which higher efﬁciency leads to higher cell kill.

Exam le 10: Immunohistochemistr IHC of mutant HSV-TK cellular rotein ex ression Either Reximmune Cl or C2 ds were transiently trasfected into 293T cells and incubated under standard condition on tissue culture slides aparatus, a couple days later cells were ﬁxed with about 2% formalin, washed with PBS and bilized with 0.1% triton x 100 or equivalent detergent. Primary anti HSV-TK antibody (Santa Cruz Biotechnology) at effective dilution is incubated with these cells 4 degrees C overnight. Cells are washed and incubated for 1-2 hours with secondary anti y antibody conjugated with horse radish peroxidase ( HRPO) at ambient room temperature. Cells are again washed and HRPO detection stain reagent is applied for 5-30 minutes at room temperature. IHC images are acquired with a light microscope ﬁtted with a CCD l camera, pictures are ed with image analysis re. Note: IHC in this example can also be described as ImmunoCyto Chemistry (ICC).

Wild type vector was found to localize to the nucleus ( As determined with ﬂuorescent genes fused to wild type HSV-TK), Data not shown..

RexCl distributes between nucleus and cytoplasm in ﬂuorescent fusion (data not shown), but mostly r in Immunohistochemistry (IHC; see, Figure 37, left panel).

ReXC2 is almost entirely cytoplasmic in ﬂuorescent fusion (data not shown), with some shift to the cytoplasm seen in IHC (see, Figure 37, right panel).

PCT/U82014/029814 Example 11: Improved s The effect of various mutations were compared to previously disclosed constructs such as those described by Margaret Black. Rescue of BL21 DE3 tk(-) Cells by HSV-TK Variant pET Constructs is shown in the ing table: ConcentrationTh midine m /mL {onsti11m W“ ----- (imNES dmNES The following table depicts GCV Kill after Rescue of BL21 DE3 tk(-) Cells by HSV-TK Variant pET ucts.

Growth aﬁe: 24 hr 3nwbaiion at 3?“; m: pTK# = pET30a-based bacterial protein expression vector encoding an HSV-TK gene or variant; pTKl = wild-type ; pTK2 = HSV-TK NESdmNLS Al67Y(SR39); pTK3 = HSV-TK(SR39) (As in Reximmune-Cl); pTK4 = HSV-TK-NESdmNLS Al67F; pTKS = HSV- TK-NESdmNLS Al68H ( As in Reximmune-CZ); pET24a = empty expression vector as negative control; GCV = ganciclovir (at the indicated concentrations), IPTG = isopropyl b-D-l- thiogalactopyranoside (as lac operon inducer for HSV-TK protein expression); 2xYT = 2x yeast/tryptone bacterial media in agar plates, where the trials in the column so labeled lack both IPTG and GCV. All of these HSV-TK’s are codon optimized for expression in prokaryotes and expressed in the IPTG inducible pET30a plasmid. Note; HSV-TK Mutants which do not have Thymidine enzymatic ty will not support the growth of these TK minus bacterial cells. e 12: In vitro Bystander Assays ] Experiments were conducted at our tory to demonstrate the bystander effect in vitro on mixtures of cancer cells expressing various TK mutants with non-expressing cells. A375 human melanoma and C6 rat glioma stable pure tion cell lines were established containing the A168H mutated HSV-TK-m2 gene. The bystander assays were conducted by plating the cancer cells with mixtures of the parental non-HSV-TK-m2 cells with the corresponding HSV-TK-m2 cell line ranging from 0-lOO% HSV-TK-m2. The mixtures of cancer cells were subsequently exposed to 5-20 uM GCV and cell kill is plotted in the ﬁgures below. The results clearly show signiﬁcant increases in the mixed populations over what would be considered tical, t a bystander effect.

More than 40 bystander assays were performed using different mutant TK and clonal populations. The data was compiled with GCV sensitivity and enzyme kinetic measurements as well as viral titer of production for the mutants with potential.

Figure 29 provides graphic results from one bystander in vitro assay for various mutants. The data support that mutated HSV-TK Al68H gene has a higher cell kill and bystander effect than the HSV-TK 167 or Margaret Black mutants.

Figure 30 provides a c from a bystander in vitro assay where C6-Hygro-TK clones were treated with 20 mM GCV. The data further support that HSV-TK Margaret Black mutants had the lowest cell kill of the other mutants tested.

Analysis of all of the mutants identified mutant TKl68dmNES to be a lead candidate for the TK gene in une C-2.

Example 13: Seguences of modiﬁed TK molecules HSV-TK Splice Sites Removal; optimized TKl [salice sites ted] ATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGCCTTCGACCAGGCCGCCCGCAG CCGCGGCCACAGCAACGGCAGCACCGCCCTGCGCCCCCGCCGCCAGCAGGAGGCCA CCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCGACGGC CCCCACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCG CGACGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCA GCGAGACCATCGCCAACATCTACACCACCCAGCACCGCCTGGACCAaGGCGAGATC AGCGCCGGCGACGCCGCCGTGGTGATGACCAGCGCCCAGATCACCATGGGCATGCC CTACGCCGTGACCGACGCCGTGCTGGCCCCCCACATCGGCGGCGAGGCCGGCAGCA GCCACGCCCCCCCCCCCGCCCTGACCATCTTCCTGGACCGCCACCCCATCGCCTTCA TGCTGTGCTACCCCGCCGCCCGCTACCTGATGGGCAGCATGACaCCaCAaGCCGTGCT GGCCTTCGTGGCCCTGATCCCCCCCACCCTGCCCGGCACCAACATCGTGCTGGGCGC CCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAGC GCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAAC CGCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAG CGGCACCGCCGTGCCCCCCCAGGGCGCCGAGCCCCAGAGCAACGCCGGCCCCCGCC CCCACATCGGCGACACCCTGTTCACCCTGTTCCGCGCCCCCGAGCTGCTGGCCCCCA ACGGCGACCTGTACAACGTGTTCGCCTGGGCCCTGGACGTGCTGGCCAAGCGCCTG CGCAGCATGCACGTGTTCATCCTGGACTACGACCAGAGCCCCGCCGGCTGCCGCGA CGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACCACCCCCGGCA GCATCCCCACCATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGGCC AACTAA ] Codon-optimized, all putative splice acceptor sites ablateda TK1 with RE’s,+Kozaka 2XTK A168H gLIF. . .AHL! gKaGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGC CAGCGCCTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCM: TIKKQCCaCGgCGCCAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCC ACCCTGCTGCGCGTGTACATCGACGGaCCaCACGGCATGGGCAAGACCACCACCACC CAGCTGCTGGTGGCCCTGGGCAGCCGCGACGACATCGTGTACGTGCCCGAGCCCAT GACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATCGCCAACATCTACACCACCC AGCACCGCCTGGACCAaGGCGAGATCAGCGCCGGCGACGCCGCCGTGGTGATGACC AGCGCCCAGAJtACaATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGCaCCa CACATCGGCGGCGAGGCCGGCAGCAGCCACGCaCCMXhCCMXhCTGACCCTGATC TTCGACCGgCACCCaATCGCaCACCTGCTGTGCTACCCgGCaGCaCGCTACCTGATGG GAﬁhCCBCAaGCCGTGCTGGCCTTCGTGGCCCTGATCCCaCCaACaCTGCCCG GCACCAACATCGTGCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCC AAGCGCCAGCGCCCCGGCGAGCGCCTGGACCTGGCCATGCTGGCCGCCATCCGCCG CGTGTACGGCCTGCTGGCCAACACCGTGCGCTACCTGCAGTGCGGCGGCAGCTGGC GCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCCthCAGGGCGCCGAGCCa CAGAGCAACGCCGGaCCaCGaCCMQACATCGGCGACACCCTGTTCACCCTGTTCCGgG CaCCaGAGCTGCTGGCaCCaAACGGCGACCTGTACAACGTGTTCGCCTGGGCCCTGG TGGCCAAGCGCCTGCGCumATGCACGTGTTCATCCTGGACTACGACCAGga CCgGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCA CGTGACaACaCCCGGCAGCATCCCaACaATCTGCGACCTGGCCCGCACCTTCGCCCGC GAGATGGGCGAGGCCAACTAATAGGGATCCCTCGAGAAGCTTga: ] HSV-TK Splice Sites Removal Improves Codon Optimization gtcaGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGC CAGCGCCTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCaC TGCGgCCaCGgCGCCAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCC ACCCTGCTGCGCGTGTACATCGACGGaCCaCACGGCATGGGCAAGACCACCACCACC CAGCTGCTGGTGGCCCTGGGCAGCCGCGACGACATCGTGTACGTGCCCGAGCCCAT GACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATCGCCAACATCTACACCACCC AGCACCGCCTGGACCAaGGCGAGATCAGCGCCGGCGACGCCGCCGTGGTGATGACC AGCGCCCAGATtACaATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGCaCCaC ACATCGGCGGCGAGGCCGGCAGCAGCCACGCaCCaCCaCCaGCaCTGACCCTGATCTT QGACCGgCACCCaATCGCaCACCTGCTGTGCTACCCgGCaGCaCGCTACCTGATGGGCt ccATGACaCCaCAaGCCGTGCTGGCCTTCGTGGCCCTGATCCCaCCaACaCTGCCCGGCA CCAACATCGTGCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAG CGCCCCGGCGAGCGCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGT GTACGGCCTGCTGGCCAACACCGTGCGCTACCTGCAGTGCGGCGGCAGCTGGCGCG AGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCCaCCaCAGGGCGCCGAGCCaCAG AGCAACGCCGGaCCaCGaCCaCACATCGGCGACACCCTGTTCACCCTGTTCCGgGCaCC aGAGCTGCTGGCaCCaAACGGCGACCTGTACAACGTGTTCGCCTGGGCCCTGGACGT GCTGGCCAAGCGCCTGCGthcATGCACGTGTTCATCCTGGACTACGACCAGtcaCCgG @GGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTG ACaACaCCCGGCAGCATCCCaACaATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGA TGGGCGAGGCCAACTAATAGGGATCCCTCGAGAAGCTTgtca HSV-TK NLS Removal and tute in NES gtcaGCGGCCGCACCGGTACGCGTCCACCﬂGCCAGCTACCCCGGCCA CCAGCACGCCAGCGCCTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCA GCACCGCaCTGCGgCCaCGgCGCCAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGA AGATGCCCACCCTGCTGCGCGTGTACATCGACGGaCCaCACGGCATGGGCAAGACCA CCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGACGACATCGTGTACGTGCCC GAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATCGCCAACATCTA CACCACCCAGCACCGCCTGGACCAaGGCGAGATCAGCGCCGGCGACGCCGCCGTGG TGATGACCAGCGCCCAGATtACaATGGGCATGCCCTACGCCGTGACCGACGCCGTGC TGGCaCCaCACATCGGCGGCGAGGCCGGCAGCAGCCACGCaCCaCCaCCaGCaCTGAC CTTCGACCGgCACCCaATCGCaCACCTGCTGTGCTACCCgGCaGCaCGCTACC TGATGGGthcATGACaCCaCAaGCCGTGCTGGCCTTCGTGGCCCTGATCCCaCCaACaC TGCCCGGCACCAACATCGTGCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGC CTGGCCAAGCGCCAGCGCCCCGGCGAGCGCCTGGACCTGGCCATGCTGGCCGCCAT CGTGTACGGCCTGCTGGCCAACACCGTGCGCTACCTGCAGTGCGGCGGCA GCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCCaCCaCAGGGCGCC GAGCCaCAGAGCAACGCCGGaCCaCGaCCaCACATCGGCGACACCCTGTTCACCCTGT TCCGgGCaCCaGAGCTGCTGGCaCCaAACGGCGACCTGTACAACGTGTTCGCCTGGGC CCTGGACGTGCTGGCCAAGCGCCTGCGthcATGCACGTGTTCATCCTGGACTACGAC CAGtcaCCgGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAG ACCCACGTGACaACaCCCGGCAGCATCCCaACaATCTGCGACCTGGCCCGCACCTTCG CCCGCGAGATGGGCGAGGCCAACTAATAGGGATCCCTCGAGAAGCTTgtca HSV-TK NLS Removal gtcaGCGGCCGCACCGGTACGCGTCCACCﬂGCCAGCTACCCCGGCCACCAGCACGC CAGCGCCTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCaC TGCGgCCaGGATCTCAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCC ACCCTGCTGCGCGTGTACATCGACGGaCCaCACGGCATGGGCAAGACCACCACCACC CAGCTGCTGGTGGCCCTGGGCAGCCGCGACGACATCGTGTACGTGCCCGAGCCCAT GACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATCGCCAACATCTACACCACCC AGCACCGCCTGGACCAaGGCGAGATCAGCGCCGGCGACGCCGCCGTGGTGATGACC AGCGCCCAGATtACaATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGCaCCaC ACATCGGCGGCGAGGCCGGCAGCAGCCACGCaCCaCCaCCaGCaCTGACCCTGATCTT QGACCGgCACCCaATCGCaCACCTGCTGTGCTACCCgGCaGCaCGCTACCTGATGGGCt ccATGACaCCaCAaGCCGTGCTGGCCTTCGTGGCCCTGATCCCaCCaACaCTGCCCGGCA CCAACATCGTGCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAG CGCCAGCGCCCCGGCGAGCGCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGT GTACGGCCTGCTGGCCAACACCGTGCGCTACCTGCAGTGCGGCGGCAGCTGGCGCG AGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCCaCCaCAGGGCGCCGAGCCaCAG AGCAACGCCGGaCCaCGaCCaCACATCGGCGACACCCTGTTCACCCTGTTCCGgGCaCC aGAGCTGCTGGCaCCaAACGGCGACCTGTACAACGTGTTCGCCTGGGCCCTGGACGT GCTGGCCAAGCGCCTGCGthcATGCACGTGTTCATCCTGGACTACGACCAGtcaCCgG EGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTG CCCGGCAGCATCCCaACaATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGA TGGGCGAGGCCAACTAATAGGGATCCCTCGAGAAGCTTgtca HSV-TK Custom Codon Optimization gtcaGCGGCCGCACCGGTACGCGTCCACCﬂGCCCTGCAGAAAAAGCTGGAAGAGC TGGAACTGGATGGCTCTTATCCT GGACATCAGCATGCTTCTGCTTTTGATCAGGCTGCCAGATCTAGAGGACATTCTAAT GGCAGCACAGCACTGCGGCCAGGATCTCAGCAGGAAGCTACAGAAGTGAGACCTG AACAGAAAATGCCTACACTGCTGAGAGTGTATATTGATGGACCACATGGAATGGGA ACCACAACCCAGCTGCTGGTGGCTCTCGGATCTAGAGATGATATTGTGTA TGTGCCTGAACCTATGACATATTGGAGAGTGCTGGGAGCTTCTGAAACAATTGCTA ATATCTATACAACACAGCATAGACTGGATCAAGGAGAAATTTCTGCCGGAGATGCT GCCGTGGTGATGACATCTGCTCAGATTACAATGGGAATGCCTTATGCTGTGACAGAT GCTGTGCTGGCACCACATATTGGAGGCGAAGCTGGAAGCTCTCATGCACCACCACC AGCACTGACACTGATTTTTGATCGGCATCCAATTGCACATCTGCTGTGTTATCCGGC AGCAAGATATCTGATGGGAAGCATGACACCACAAGCCGTGCTGGCTTTTGTGGCTC TGATTCCACCAACACTGCCTGGAACAAACATCGTGCTGGGAGCTCTGCCTGAAGAT AGACATATCGATCGGCTGGCCAAACGGCAGAGACCTGGAGAACGGCTGGATCTGGC CATGCTGGCTGCCATTCGGAGAGTGTATGGCCTGCTGGCTAACACAGTGAGATATCT GCAGTGTGGAGGCTCTTGGAGAGAGGATTGGGGACAGCTGTCTGGCACAGCTGTGC CACCACAGGGAGCCGAACCACAGAGCAATGCTGGACCACGACCACATATCGGAGA CACACTGTTTACACTGTTTCGGGCACCAGAACTGCTGGCACCAAATGGAGACCTGT ACAACGTGTTTGCCTGGGCTCTGGATGTGCTGGCTAAACGGCTGAGATCTATGCATG TGTTTATCCTGGACTATGATCAGTCACCGGCCGGATGTCGCGATGCCCTGCTGCAGC CTGGGATGGTGCAGACACATGTGACAACACCTGGATCTATCCCAACAATC TGTGATCTGGCTAGAACATTCGCTAGGGAGATGGGAGAGGCCAACTAATAGGGATC CCTCGAGAAGCTTgwa HSV-TK NLS Removal NES and Addition gKmGCGGCCGCACCGGTACGCGTCCACCAIQGCCCTGCAGAAAAAGCTGGAAGAGC TGGATGGCAGCTACCCCGGCCACCAGCACGCCAGCGCCTTCGACCAGGCC GCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCﬂHKKXhﬁKhGGATCTCAGCAG GAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACAT CGACGGﬂXhCACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGG GCAGCCGCGACGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTG GGCGCCAGCGAGACCATCGCCAACATCTACACCACCCAGCACCGCCTGGACCAaGG CGAGATCAGCGCCGGCGACGCCGCCGTGGTGATGACCAGCGCCCAGATUKhATGGG CATGCCCTACGCCGTGACCGACGCCGTGCTGGCﬂXhCACATCGGCGGCGAGGCCGG CAGCAGCCALIKhCCkaKKhGCaCTGACCCTGATCTTCGACCGgCACCCaATCGCaC ACCTGCTGTGCTACCCgGCaGCaCGCTACCTGATGGGCKmATGACaCCaCAaGCCGTGC TGGCCTTCGTGGCCCTGATCCCﬂXhAﬁhCTGCCCGGCACCAACATCGTGCTGGGCGC CCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAGC GCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAAC CGCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAG CGGCACCGCCGTGCCﬂXhCAGGGCGCCGA£KKhCAGAGCAACGCCGGﬂXhCGﬂXh CACATCGGCGACACCCTGTTCACCCTGTHXXQGCﬂXhGAGCTGCTGGCﬂXhAACG GCGACCTGTACAACGTGTTCGCCTGGGCCCTGGACGTGCTGGCCAAGCGCCTGCGCt WATGCACGTGTTCATCCTGGACTACGACCAGwﬂXgGCCGGCTGCCGCGACGCCCTG CTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACMUhCCCGGCAGCATCCCa ACaATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGGCCAACTAATA GGGATCCCTCGAGAAGCTTgwa HSV-TK Custom Codon zation gKmGCGGCCGCACCGGTACGCGTCCACCAIQGCCCTGCAGAAAAAGCTGGAAGAGC TGGAACTGGATGGCTCTTATCCT GGACATCAGCATGCTTCTGCTTTTGATCAGGCTGCCAGATCTAGAGGACATTCTAAT ACAGCACTGCGGCCAGGATCTCAGCAGGAAGCTACAGAAGTGAGACCTG AACAGAAAATGCCTACACTGCTGAGAGTGTATATTGATGGACCACATGGAATGGGA AAAACAACCACAACCCAGCTGCTGGTGGCTCTCGGATCTAGAGATGATATTGTGTA TGTGCCTGAACCTATGACATATTGGAGAGTGCTGGGAGCTTCTGAAACAATTGCTA ATATCTATACAACACAGCATAGACTGGATCAAGGAGAAATTTCTGCCGGAGATGCT GCCGTGGTGATGACATCTGCTCAGATTACAATGGGAATGCCTTATGCTGTGACAGAT GCTGTGCTGGCACCACATATTGGAGGCGAAGCTGGAAGCTCTCATGCACCACCACC AGCACTGACACTGATTTTTGATCGGCATCCAATTGCACATCTGCTGTGTTATCCGGC AGCAAGATATCTGATGGGAAGCATGACACCACAAGCCGTGCTGGCTTTTGTGGCTC TGATTCCACCAACACTGCCTGGAACAAACATCGTGCTGGGAGCTCTGCCTGAAGAT AGACATATCGATCGGCTGGCCAAACGGCAGAGACCTGGAGAACGGCTGGATCTGGC CATGCTGGCTGCCATTCGGAGAGTGTATGGCCTGCTGGCTAACACAGTGAGATATCT GCAGTGTGGAGGCTCTTGGAGAGAGGATTGGGGACAGCTGTCTGGCACAGCTGTGC CACCACAGGGAGCCGAACCACAGAGCAATGCTGGACCACGACCACATATCGGAGA CACACTGTTTACACTGTTTCGGGCACCAGAACTGCTGGCACCAAATGGAGACCTGT ACAACGTGTTTGCCTGGGCTCTGGATGTGCTGGCTAAACGGCTGAGATCTATGCATG TGTTTATCCTGGACTATGATCAGTCACCGGCCGGATGTCGCGATGCCCTGCTGCAGC TGACATCTGGGATGGTGCAGACACATGTGACAACACCTGGATCTATCCCAACAATC TGTGATCTGGCTAGAACATTCGCTAGGGAGATGGGAGAGGCCAACTAATAGGGATC CCTCGAGAAGCTTgwa WO 53258 Example 14: HAT Assay Retroviral Vectors of RexRed Super TK Al68H and RexRed TK 167F were produced in 293T cells and used to transduce 3T3 (TK-) cells. These transduced cells were HAT selected for 7-14 days. Untransduced 3T3 (TK-) cells will die post HAT selection. These same cells transduced with RexRed Super TK Al68H did survive HAT selection, however 3T3(TK-) cells transduced with RexRed TK 167F did not survive HAT selction. This is a plus/ minus cell survival assay, surviving cells are ﬁxed and stained with 1% methylene blue in methanol.

Previous transduction based HAT cell kill assays reveal a GCV speciﬁcity over thymidine for the Al67F HSV-TK mutants in retroviral vectors ning the RFP marker.

That speciﬁcity is found in NIH 3T3 cells in a 72 hour and 7 day assay at lx HAT dose.

Current transduction based HAT cell kill assays reveal a GCV speciﬁcity over thymidine for the Al67F HSV-TK s in retroviral s containing the RFP marker.

That speciﬁcity is found in NIH 3T3 cells in a 7 day assay at 2x HAT dose.

Transduction based HAT cell kill assays reveal a GCV speciﬁcity over thymidine for the Al67F HSV-TK mutants in retroviral vectors containing the RFP marker. That speciﬁcity is found in NIH 3T3 cells in a 72 hour assay and 7 day assay at lx HAT dose.

] Transduction based HAT cell kill assays reveal a GCV speciﬁcity over thymidine for the Al67F HSV-TK mutants in retroviral vectors containing the HygroR marker. That speciﬁcity is found in NIH 3T3 cells in a 72 hour and 7 day assay at lx HAT dose.

Example 15: GCV Kill Assay Cells were seeded in a 24 well dish. Cells were transduced the next day with 6 ons of the retroviral vectors (1:4-4096). The next day 0-200 uM GCV was added to the cells. After seven days of GCV treatment the cells were ﬁxed and the live cells stain with 1% methylene blue in methanol. The higher the potency of the viral mutants leads to more cell kill.

Previous transduction based HSV-TK/GCV cell kill assays reveal a y order for Al68F, Al67F and Al68H HSV-TK mutants in retroviral s containing the RFP . That order is Al68H > Al68F = Al67F when tested in RgA375 cells in a 72 hour and 7 day assay at high GCV dose ( 1 mM — 125 mM).

Current transduction based HSV-TK/GCV cell kill assays reveal a potency order for Al67F and Al68H HSV-TK mutants in iral vectors containing the RFP marker. That order is Al68H > Al67F when tested in RgA375 cells in a 7 day assay at high GCV dose (0.2mM — 0.05mM). The addition of dm NLS or NES does not appear to change this order. The use of JCO does appear to lower titer and aggregate HSV-TK cell kill activity.

Transduction based HSV-TK/GCV cell kill assays reveal a potency order for Al68F, Al67F and Al68H HSV-TK mutants in retroviral vectors containing the RFP marker.

That order is Al68H > Al68F = Al67F when tested in A375 and RgA375 cells in a 72 hour assay at high GCV dose ( 1 mM — 125 mM).

Transduction based /GCV cell kill assays reveal a potency order for Al68F, Al67F and Al68H HSV-TK mutants in retroviral s containing the RFP marker.

That order is Al68H > Al68F = Al67F when tested in NIH 3T3 cells in a 72 hour assay at high GCV dose ( 1 mM — 500 mM).

That order is Al68H > Al68F = Al67F when tested in RgA375 cells in a 72 hour and 7 day assay at high GCV dose ( 1 mM — 125 mM).

Transduction based HSV-TK/GCV cell kill assays reveal a potency order for Al68F, Al67F and Al68H HSV-TK mutants in retroviral vectors containing the RFP or HygroR marker. That order is Al68H > Al68F = Al67F when tested in A375, RgA375 or NIH 3T3 cells in a 72 hour assay at high GCV dose ( 1 mM — 125 mM).

Transduction based HSV-TK/GCV cell kill assays reveal a potency order for Al68F, Al67F and Al68H HSV-TK mutants in retroviral vectors containing the HygroR marker. That order is Al68H > Al68F = Al67F when tested in A375 and RgA375 cells in a 72 hour assay at high GCV dose ( 1 mM — 125 mM).

] Transduction based HSV-TK/GCV cell kill assays reveal a potency order for Al68F, Al67F and Al68H HSV-TK mutants in retroviral vectors containing the HygroR marker. That order is Al68H > Al68F = Al67F when tested in NIH 3T3 cells in a 72 hour assay at high GCV dose ( 1 mM — 500 mM).

Example 16: Hygro Resistance [0051 1] Cell lines transduced with retrovector Hygro-HSV-TK mutants were selected in the ce of ycin to e a pure population of cells containg the Hygro-HSV-TK mutants and expressing the hygromycin resistence gene.

A375 une-C2 like Cell lines: A375 Hygro selected HSV-TK dmNESAl68H cell lines have been converted to Luc(+). The above cell line has same GCV kill as parental line. A A375 Luc(+) only cell line has same Luc activity as above cell line.

C6 Reximmune-C2 like Cell lines: C6 Hygro selected HSV-TK dmNESAl68H cell lines have been converted to Luc(+). The above cell line has same GCV kill as parental line.

A C6 Luc(+) only cell line has same Luc activity as above cell line. 2014/029814 While preferred embodiments have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only.

Numerous variations, changes, and substitutions will now occur to those d in the art without departing from the disclosed embodiments. It should be understood that various alternatives to the embodiments described herein may be employed in practicing the embodiments. It is intended that the following claims deﬁne the scope of the embodiments and that s and structures within the scope of these claims and their equivalent be covered thereby. 2014/029814 ABBREVIATIONS ALT Alanine aminotransferase ANC Absolute neutrophil count AST ate aminotransferase AUC Area under the plasma concentration-time curve BSA Body surface area (mg/m2) CL Systemic plasma clearance Cmax Peak plasma concentration CR Complete response CRF Case report form CT Computerized tomography CTC Common Toxicity Criteria DLT Dose Limiting Toxicities EOI End of infusion FDA Food and Drug Administration G-CSF Granulocyte-colony stimulating factor (filgrastim, Neupogen®) GCP Good al practice GM-CSF ocyte-macrophage colony-stimulating factor (sargramostim, Leukine®) HIV Human Immunodeﬁciency Virus HR Hazard ratio IEC Independent Ethics Committee i.p. 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Ther. 4: 146-8 (2001). -lO2- SE UENCES SEQ ID NO: 1: wild type HSVl-TK nucleotide sequence atggcttcgtaccccggccatcaacacgcgtctgcgttcgaccaggctgcgcgttctcgcggccatagcaaccgacg tacggcgttgcgccctcgccggcagcaagaagccacggaagtccgcccggagcagaaaatgcccacgctactgcggg tttatatagacggtccccacgggatggggaaaaccaccaccacgcaactgctggtggccctgggttcgcgcgacgat atcgtctacgtacccgagccgatgacttactggcgggtgctgggggcttccgagacaatcgcgaacatctacaccac acaacaccgcctcgaccagggtgagatatcggccggggacgcggcggtggtaatgacaagcgcccagataacaatgg gcatgccttatgccgtgaccgacgccgttctggctcctcatatcgggggggaggctgggagctcacatgccccgccc ccggccctcaccctcatcttcgaccgccatcccatcgccgccctcctgtgctacccggccgcgcggtaccttatggg cagcatgaccccccaggccgtgctggcgttcgtggccctcatcccgccgaccttgcccggcaccaacatcgtgcttg gggcccttccggaggacagacacatcgaccgcctggccaaacgccagcgccccggcgagcggctggacctggctatg ctggctgcgattcgccgcgtttacgggctacttgccaatacggtgcggtatctgcagtgcggcgggtcgtggcggga ggactggggacagctttcggggacggccgtgccgccccagggtgccgagccccagagcaacgcgggcccacgacccc atatcggggacacgttatttaccctgtttcgggcccccgagttgctggcccccaacggcgacctgtataacgtgttt gcctgggccttggacgtcttggccaaacgcctccgttccatgcacgtctttatcctggattacgaccaatcgcccgc cggctgccgggacgccctgctgcaacttacctccgggatggtccagacccacgtcaccacccccggctccataccga cgatatgcgacctggcgcgcacgtttgcccgggagatgggggaggctaactga SEQ ID NO: 2: wild type HSVl-TK amino acid ce MASYPGHQ{ASAFDQAARSRGHSNRRTALR?RRQQnATnVRPnQKMPTLLRVYIDGPHGMGKTTTTQLLVALGSRDD < ?EPMTYWRVLGASETIANIYTTQHR.DQGEISAGDAAVVMTSAQITMG ?YAVTDAVLAPHIGGEAGSSHAPPPAL H SI W DRHPIAASLCYPAARYLMGSMTPQAVLAFVASIP?TLPGTNIVLGA.P _*J DRHIDRDAKRQRPG .RLDLAM.AAIRR*J <._<G bSANTVRYSQCGGSWREDWGQLSGTAVPPQGAEPQSNAGPRPHIGDT.FT.FRAP?.LAPNGDLYVVFAWASDVLAK SMHVFILDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTEARnMGnAV SEQ ID NO: 3: HSV-TK in Reximmune-C HSV-TK; SR 39 mutant and 26S Mutation of NLS atggcctcgtaccccggcca:caacacgcgtctgcgttcgaccaggctgcgcgttctcgcggccatagca acggatccacggcg,,gcgccc:cgccggcagcaagaagccacggaag:ccgcccggagcagaaaatgcc cacgc,ac,gcggg,,La,a,agacgg:ccccacgggatggggaaaaccaccaccacgcaacLgc,gg,g gcchggg,,cgcgcgacga,a,chc,achacccgagccga,gacL,achgcgggtgctgggggce, ccgagacaatcgcgaaca,c,acaccacacaacaccgcctcgaccagggtgagatatcggccggggacgc ggcggegg,aa,gacaagcgcccagataacaatgggca,gcc,,a,gccgLgaccgacgccgttctggc: cceca,a,cgggggggaggc:gggagctcaca:gccccgcccccggccc:caccatcttcctcgaccgcc chcc,LcaLchg,chacccggccgcgcgg,acc,,a,gggcagca:gaccccccaggccg: gceggcg,ch,ggccctca:cccgccgacce,gcccggcaccaacach,ch,ggggccc:tccggag gacagacacatcgaccgcctggccaaacgccagcgccccggcgagcggctggacctggc:atgctggctg cgattcgccgcgtttacgggc,ac,,gccaaeacgngcggLaLchcag,gcggcggg,cg,ggcggga ggac:ggggacagc:ttcggggacggccgtgccgccccagggtgccgagccccagagcaacgcgggccca cgaccccatatcggggacacg,La,,Laccceg,Lchggcccccgag,,gceggcccccaacggcgacc LgLa,aachgLL,gccnggccttggacgtcttggccaaacgcctccgttccatgcacgLcEL,aLch ggat:acgaccaatcgcccgccggc:gccgggacgcccLgcLgcaacL,acceccgggatggtccagacc cacg:caccacccccggctccataccgacgatatgcgacctggcgcgcacgtttgcccgggagatggggg actga SEQ ID NO: 4 (amino acid sequence encoded by SEQ ID NO: 3) MASYPGHQHASAFDQAARSRGHSNGSTALRPRRQQnATnVRPnQKMPTLLRVY:DGPHGMGKTTTTQL;V D VYVPnPMTYWRVLGASnT AN YTTQHRLDQGn SAGDAAVVMTSAQITMGMPYAVTDAVLA VV()2014/153258 PCT/L S2014/029814 PH GG ﬂAGSSHAPPPA' J hLDRiP AhMLCYPAARYLMGSMTPQAV' .AFVAL" PPT' .PGTN"VLGALP:‘J DRH: DRLAKRQRPGTRH DLA VYGLIJANTVRYLQCGGSWRE.|. DWGQLSGTAVPPQGAEPQSNAGP RPH G DTLhTLh?AP*CF .AP GIJYNVFAWA' 'Q" .RSMHVF'". DYDQSPAGCR DALLQLTSGMVQT {VTTPGS PT CDLARTEARLMGLAN SEQ ID NO: 5: HSV-TK Sites to mutate are in bold, underlining K nuclear localization sequence, RR, and Substrate Binding Domain, LIF and AAL atggcctcgtaccccggccatcaacacgcgtc:gcgttcgaccaggctgcgcgttctcgc 60 M A S Y P G H Q i A S A F D Q A A R S R ggccatagcaaccgacgtacggcg, ,gcgccthgccggcagcaagaagccacggaag:c L20 G H S NBBT A .4 A P 5 5 Q Q :3 A T :3 v cgcccggagcagaaaatgcccacgc ,acegcggg,LLa,aLagacggtccccacgggatg L80 R P '-T‘ .L Q K M P T H H R V Y i D G P H G M accaccaccacgcaacLgc ,gg,ggccc,ggg,ecgcgcgacgatatcgtctac 240 G K T T T T Q " . H V A L G S R D D V Y gtacccgagccgatgacttac:ggcggg :gctgggggc,,ccgagacaatcgcgaacatc 300 V P r L P M T Y W R V L G A S E T A N tacaccacacaacaccgcctcgaccagggtgagatatcggccggggacgcggcggegg,a 360 Y T T Q H R L D Q G A S A G D A A V V atgacaagcgcccagataacaatgggcatgcc:tatgccgtgaccgacgccgttc:ggct M T S A Q T M G M P Y A V T D A V L A cctcatatcgggggggaggctgggagctcaca:gccccgcccccggccctcaccctcatc P i " G G E A G S S H A P P P A L T E E EEggaccgccatcccatcgccgccctcc:gtgctacccggccgcgcgg,acc,Langgc 540 E 3 R H P A A E :4 C Y P A A R Y :4 M G agcatgaccccccaggcchgc,ggcg,,chggccctcatcccgccgacc,egcccggc 600 S T P Q A V L A F V A L P P T L P G accaacatcgtgcttggggccCttccggaggacagacaca:cgaccgcctggccaaacgc 660 T " V L G A L P E D R H D R L A < R cagcgccccggcgagcggctggacc,ggcLaLchgchgcgaLchccgcgtttacggg 720 Q R P G T. R '. D L A M L A A I R R V Y G cLac,egccaaLacgngcggeaLc,gcagLgcggcgggtcgtggcgggaggaCtgggga 780 L L A N T V R Y L Q C G G S W R E D W G cagc,,Lcggggacggccgtgccgccccagggtgccgagccccagagcaacgcgggccca 840 Q L S G T A V P P Q G A E P Q S A G P cgaccccatatcggggacacge,aLLLacchg,eLcgggcccccgagttgctggccccc 900 R P i G D T L F T L F R A P H H H A P gacc,g,a,aacg,ge,,gchgggcc,eggacgtCttggccaaacgcctccgt 960 G D L Y N V F A W A L D V L A < R L R :ccatgcacgec,L,aLcc,gga,Lacgaccaa:cgcccgccggctgccgggacgccctg 1020 S M i V F L D Y D Q S P A G C R D A L c,gcaacLLacc,ccggga,gg,ccagacccacg:caccacccccggc:ccataccgacg 1080 L Q L T S G M V Q T i V T T P G S P T atatgcgacctggcgcgcacgt:tgcccgggaga:gggggaggctaac:ga C D L A R T F A R E M G E A N * SEQ ID NOS: 6 and 7: Sac I-Kpn I (SR39) mutant region GAGCTCACATGCCCCGCCCCCGGCCCTCACCéTCETCETCGACCGCCATCCCATCGCC- ETCGAGTGTACGGGGCGGGGGCCGGGAGTGGEAGéAGEAGCTGGCGGTAGGGTAGCGG- —104— 2014/029814 -EEC§T§CTGTGCTACCCGGCCGCGCGGTACE (SﬁQ 3 NO: 6) —A_AGEAEGACACGATGGGCCGGCGCGCCATGG (S *1Q 3 NO: 7) Kpn " IIIIIIIIIII - 3’ IIIII "III - 3’ GAGCTC IIIIIIIIIII IIIIIIIIIII SEQ ID NOS: 8 and 9: Sac I-Kpn I (SR39) mutant region (cut) CACATGCCCCGCCCCCGGCCCTCACCéTCETCETCGACCGCCATCCCATCGCCEECATE GTACGGGGCGGGGGCCGGGAGTGGEAGQAGEAGCTGGCGGTAGGGTAGCGGAE Sac I (cut) CTGTGCTACCCGGCCGCGCGGTAC (SﬁQ 3 NO: 8) GEAEGACACGATGGGCCGGC (SﬁQ 3 NO: 9) Kpn I(Cut) GGTACC IIIIIIIIIII GTAC - 3’ IIIIIIIIIII SEQ ID NOS: 10 and 11: Primers SR39sackpn Fl 'CACATGCCCCGCCCCCGGCCCTCACCETCETCETCGACCGCCATCCCATCGCCTTCATGCTGTGCTAC CCGGCCGCGCGGTAC 3’ (SﬁQ 3 NO: 10) SR39sackpn R1 ’CGCGCGGCCGGGTAGCACAGCATGAAGGCGATGGGATGGCGGTCGAEGAAGAEGGTGAGGGCCGGGGG ATGTGAGCT 3' (SﬁQ 3 NO: 11) SEQ ID NO: 12 Gene #3 mHSV-TK CO A168H(LIF...AHL): Length:1185 GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGC CTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCACTGCGGCCACGGCGC CAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG ACGGACCACACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA CGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC GCCAACATCTACACCACCCAGCACCGCCTGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCG TGGTGATGACCAGCGCCCAGATTACAATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGC ACCACACATCGGCGGCGAGGCCGGCAGCAGCCACGCACCACCACCAGCACTGACCCTGATCTTC GACCGGCACCCAATCGCACACCTGCTGTGCTACCCGGCAGCACGCTACCTGATGGGCTCCATGA CACCACAAGCCGTGCTGGCCTTCGTGGCCCTGATCCCACCAACACTGCCCGGCACCAACATCGT CGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAG CGCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGC GCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCC ACCACAGGGCGCCGAGCCACAGAGCAACGCCGGACCACGACCACACATCGGCGACACCCTGTTC ACCCTGTTCCGGGCACCAGAGCTGCTGGCACCAAACGGCGACCTGTACAACGTGTTCGCCTGGG CCCTGGACGTGCTGGCCAAGCGCCTGCGCTCCATGCACGTGTTCATCCTGGACTACGACCAGTC ACCGGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACA ACACCCGGCAGCATCCCAACAATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGG CCAACTAATAGGGATCCCTCGAGAAGCTTGTCA SEQ ID NO: 13 Gene #4 mHSV-TK CO TK A167F(LIF...FAL): :1185 GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGC CTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCACTGCGGCCACGGCGC CAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG ACGGACCACACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA CGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC GCCAACATCTACACCACCCAGCACCGCCTGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCG TGGTGATGACCAGCGCCCAGATTACAATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGC ACCACACATCGGCGGCGAGGCCGGCAGCAGCCACGCACCACCACCAGCACTGACCCTGATCTTC GACCGGCACCCAATCTTCGCACTGCTGTGCTACCCGGCAGCACGCTACCTGATGGGCTCCATGA AAGCCGTGCTGGCCTTCGTGGCCCTGATCCCACCAACACTGCCCGGCACCAACATCGT GCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAG GACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGC GCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCC ACCACAGGGCGCCGAGCCACAGAGCAACGCCGGACCACGACCACACATCGGCGACACCCTGTTC ACCCTGTTCCGGGCACCAGAGCTGCTGGCACCAAACGGCGACCTGTACAACGTGTTCGCCTGGG CCCTGGACGTGCTGGCCAAGCGCCTGCGCTCCATGCACGTGTTCATCCTGGACTACGACCAGTC ACCGGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACA GGCAGCATCCCAACAATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGG CCAACTAATAGGGATCCCTCGAGAAGCTTGTCA SEQ ID NO: 14 Gene #5 mHSV-TK CO dual mutant A167F-A168H (LIF...FHL): Length:1185 GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGC CTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCACTGCGGCCACGGCGC CAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG ACGGACCACACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA CGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC GCCAACATCTACACCACCCAGCACCGCCTGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCG TGGTGATGACCAGCGCCCAGATTACAATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGC ACCACACATCGGCGGCGAGGCCGGCAGCAGCCACGCACCACCACCAGCACTGACCCTGATCTTC GACCGGCACCCAATCTTCCACCTGCTGTGCTACCCGGCAGCACGCTACCTGATGGGCTCCATGA CACCACAAGCCGTGCTGGCCTTCGTGGCCCTGATCCCACCAACACTGCCCGGCACCAACATCGT GCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAG CGCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGC TGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCC ACCACAGGGCGCCGAGCCACAGAGCAACGCCGGACCACGACCACACATCGGCGACACCCTGTTC TTCCGGGCACCAGAGCTGCTGGCACCAAACGGCGACCTGTACAACGTGTTCGCCTGGG CCCTGGACGTGCTGGCCAAGCGCCTGCGCTCCATGCACGTGTTCATCCTGGACTACGACCAGTC ACCGGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACA GGCAGCATCCCAACAATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGG CCAACTAATAGGGATCCCTCGAGAAGCTTGTCA SEQ ID NO: 15 Gene #6 mHSV-TK CO MB-IFL A168H(IFL...AHL): Length:1185 GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGC CTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCACTGCGGCCACGGCGC CAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG ACGGACCACACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA CGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC GCCAACATCTACACCACCCAGCACCGCCTGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCG TGGTGATGACCAGCGCCCAGATTACAATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGC ACCACACATCGGCGGCGAGGCCGGCAGCAGCCACGCACCACCACCAGCACTGACCATCTTCCTG GACCGGCACCCAATCGCACACCTGCTGTGCTACCCGGCAGCACGCTACCTGATGGGCTCCATGA CACCACAAGCCGTGCTGGCCTTCGTGGCCCTGATCCCACCAACACTGCCCGGCACCAACATCGT GCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAG CGCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGC GCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCC ACCACAGGGCGCCGAGCCACAGAGCAACGCCGGACCACGACCACACATCGGCGACACCCTGTTC ACCCTGTTCCGGGCACCAGAGCTGCTGGCACCAAACGGCGACCTGTACAACGTGTTCGCCTGGG CCCTGGACGTGCTGGCCAAGCGCCTGCGCTCCATGCACGTGTTCATCCTGGACTACGACCAGTC ACCGGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACA ACACCCGGCAGCATCCCAACAATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGG CCAACTAATAGGGATCCCTCGAGAAGCTTGTCA SEQ ID NO: 16 Gene #1 HSV-TK A168H dmNLS CO SC: Length:1185 GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGC CTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCACTGCGGCCAGGATCT CAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG CACACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA CGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC GCCAACATCTACACCACCCAGCACCGCCTGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCG TGGTGATGACCAGCGCCCAGATTACAATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGC ACCACACATCGGCGGCGAGGCCGGCAGCAGCCACGCACCACCACCAGCACTGACCCTGATCTTC GACCGGCACCCAATCGCACACCTGCTGTGCTACCCGGCAGCACGCTACCTGATGGGCTCCATGA CACCACAAGCCGTGCTGGCCTTCGTGGCCCTGATCCCACCAACACTGCCCGGCACCAACATCGT GCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAG CGCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGC GCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCC ACCACAGGGCGCCGAGCCACAGAGCAACGCCGGACCACGACCACACATCGGCGACACCCTGTTC ACCCTGTTCCGGGCACCAGAGCTGCTGGCACCAAACGGCGACCTGTACAACGTGTTCGCCTGGG CCCTGGACGTGCTGGCCAAGCGCCTGCGCTCCATGCACGTGTTCATCCTGGACTACGACCAGTC ACCGGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACA ACACCCGGCAGCATCCCAACAATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGG CCAACTAATAGGGATCCCTCGAGAAGCTTGTCA SEQ ID NO: 17 Gene #2 HSV-TK A167F dmNLS CO SC: Length:1185 GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGC CTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCACTGCGGCCAGGATCT GAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG ACGGACCACACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA CGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC GCCAACATCTACACCACCCAGCACCGCCTGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCG TGGTGATGACCAGCGCCCAGATTACAATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGC ACCACACATCGGCGGCGAGGCCGGCAGCAGCCACGCACCACCACCAGCACTGACCCTGATCTTC CACCCAATCTTCGCACTGCTGTGCTACCCGGCAGCACGCTACCTGATGGGCTCCATGA CACCACAAGCCGTGCTGGCCTTCGTGGCCCTGATCCCACCAACACTGCCCGGCACCAACATCGT GCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAG GACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGC GCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCC ACCACAGGGCGCCGAGCCACAGAGCAACGCCGGACCACGACCACACATCGGCGACACCCTGTTC TTCCGGGCACCAGAGCTGCTGGCACCAAACGGCGACCTGTACAACGTGTTCGCCTGGG CCCTGGACGTGCTGGCCAAGCGCCTGCGCTCCATGCACGTGTTCATCCTGGACTACGACCAGTC ACCGGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACA ACACCCGGCAGCATCCCAACAATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGG CCAACTAATAGGGATCCCTCGAGAAGCTTGTCA SEQ ID NO: 18 Gene #3 HSV-TK A168H NESdmNLS CO SC: Length:1221 GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCCTGCAGAAAAAGCTGGAAGAGCTGGAACT GGATGGCAGCTACCCCGGCCACCAGCACGCCAGCGCCTTCGACCAGGCCGCCCGCAGCCGCGGC CACAGCAACGGCAGCACCGCACTGCGGCCAGGATCTCAGCAGGAGGCCACCGAGGTGCGCCCCG AGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCGACGGACCACACGGCATGGGCAAGACCAC CACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGACGACATCGTGTACGTGCCCGAGCCCATG ACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATCGCCAACATCTACACCACCCAGCACCGCC TGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCGTGGTGATGACCAGCGCCCAGATTACAAT GGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGCACCACACATCGGCGGCGAGGCCGGCAGC AGCCACGCACCACCACCAGCACTGACCCTGATCTTCGACCGGCACCCAATCGCACACCTGCTGT GCTACCCGGCAGCACGCTACCTGATGGGCTCCATGACACCACAAGCCGTGCTGGCCTTCGTGGC CCTGATCCCACCAACACTGCCCGGCACCAACATCGTGCTGGGCGCCCTGCCCGAGGACCGCCAC ATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAGCGCCTGGACCTGGCCATGCTGGCCGCCA TCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGCGCTACCTGCAGTGCGGCGGCAGCTGGCG CGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCCACCACAGGGCGCCGAGCCACAGAGCAAC GCCGGACCACGACCACACATCGGCGACACCCTGTTCACCCTGTTCCGGGCACCAGAGCTGCTGG CACCAAACGGCGACCTGTACAACGTGTTCGCCTGGGCCCTGGACGTGCTGGCCAAGCGCCTGCG CTCCATGCACGTGTTCATCCTGGACTACGACCAGTCACCGGCCGGCTGCCGCGACGCCCTGCTG CAGCTGACCAGCGGCATGGTGCAGACCCACGTGACAACACCCGGCAGCATCCCAACAATCTGCG ACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGGCCAACTAATAGGGATCCCTCGAGAAGCT TGTCA 2014/029814 SEQ ID NO: 19 Gene #4 HSV-TK A167F NESdmNLS CO SC: Length:1221 GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCCTGCAGAAAAAGCTGGAAGAGCTGGAACT GGATGGCAGCTACCCCGGCCACCAGCACGCCAGCGCCTTCGACCAGGCCGCCCGCAGCCGCGGC CACAGCAACGGCAGCACCGCACTGCGGCCAGGATCTCAGCAGGAGGCCACCGAGGTGCGCCCCG AGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCGACGGACCACACGGCATGGGCAAGACCAC CACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGACGACATCGTGTACGTGCCCGAGCCCATG ACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATCGCCAACATCTACACCACCCAGCACCGCC TGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCGTGGTGATGACCAGCGCCCAGATTACAAT GGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGCACCACACATCGGCGGCGAGGCCGGCAGC AGCCACGCACCACCACCAGCACTGACCCTGATCTTCGACCGGCACCCAATCTTCGCACTGCTGT GCTACCCGGCAGCACGCTACCTGATGGGCTCCATGACACCACAAGCCGTGCTGGCCTTCGTGGC CCTGATCCCACCAACACTGCCCGGCACCAACATCGTGCTGGGCGCCCTGCCCGAGGACCGCCAC CGCCTGGCCAAGCGCCAGCGCCCCGGCGAGCGCCTGGACCTGGCCATGCTGGCCGCCA TCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGCGCTACCTGCAGTGCGGCGGCAGCTGGCG CGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCCACCACAGGGCGCCGAGCCACAGAGCAAC GCCGGACCACGACCACACATCGGCGACACCCTGTTCACCCTGTTCCGGGCACCAGAGCTGCTGG CACCAAACGGCGACCTGTACAACGTGTTCGCCTGGGCCCTGGACGTGCTGGCCAAGCGCCTGCG CTCCATGCACGTGTTCATCCTGGACTACGACCAGTCACCGGCCGGCTGCCGCGACGCCCTGCTG CAGCTGACCAGCGGCATGGTGCAGACCCACGTGACAACACCCGGCAGCATCCCAACAATCTGCG ACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGGCCAACTAATAGGGATCCCTCGAGAAGCT TGTCA SEQ ID NO: 20 Gene #5 HSV-TK A168H NESdmNLS JCO SC: Length:1221 GGCCGCACCGGTACGCGTCCACCATGGCTCTGCAGAAAAAGCTGGAAGAGCTGGAACT GGATGGCTCTTATCCTGGACATCAGCATGCTTCTGCTTTTGATCAGGCTGCCAGATCTAGAGGA CATTCTAATGGCAGCACAGCACTGCGGCCAGGATCTCAGCAGGAAGCTACAGAAGTGAGACCTG AACAGAAAATGCCTACACTGCTGAGAGTGTATATTGATGGACCACATGGAATGGGAAAAACAAC CACAACCCAGCTGCTGGTGGCTCTCGGATCTAGAGATGATATTGTGTATGTGCCTGAACCTATG ACATATTGGAGAGTGCTGGGAGCTTCTGAAACAATTGCTAATATCTATACAACACAGCATAGAC TGGATCAAGGAGAAATTTCTGCCGGAGATGCTGCCGTGGTGATGACATCTGCTCAGATTACAAT GGGAATGCCTTATGCTGTGACAGATGCTGTGCTGGCACCACATATTGGAGGCGAAGCTGGAAGC TCTCATGCACCACCACCAGCACTGACACTGATTTTTGATCGGCATCCAATTGCACATCTGCTGT GTTATCCGGCAGCAAGATATCTGATGGGAAGCATGACACCACAAGCCGTGCTGGCTTTTGTGGC TCTGATTCCACCAACACTGCCTGGAACAAACATCGTGCTGGGAGCTCTGCCTGAAGATAGACAT ATCGATCGGCTGGCCAAACGGCAGAGACCTGGAGAACGGCTGGATCTGGCCATGCTGGCTGCCA TTCGGAGAGTGTATGGCCTGCTGGCTAACACAGTGAGATATCTGCAGTGTGGAGGCTCTTGGAG AGAGGATTGGGGACAGCTGTCTGGCACAGCTGTGCCACCACAGGGAGCCGAACCACAGAGCAAT GCTGGACCACGACCACATATCGGAGACACACTGTTTACACTGTTTCGGGCACCAGAACTGCTGG CACCAAATGGAGACCTGTACAACGTGTTTGCCTGGGCTCTGGATGTGCTGGCTAAACGGCTGAG GCATGTGTTTATCCTGGACTATGATCAGTCACCGGCCGGATGTCGCGATGCCCTGCTG CAGCTGACATCTGGGATGGTGCAGACACATGTGACAACACCTGGATCTATCCCAACAATCTGTG ATCTGGCTAGAACATTCGCTAGGGAGATGGGAGAGGCCAACTAATGAGGATCCCTCGAGAAGCT TGTCA SEQ ID NO: 21 Gene #6 HSV-TK A167F NESdmNLS JCO SC: Length:1221 GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCTCTGCAGAAAAAGCTGGAAGAGCTGGAACT GGATGGCTCTTATCCTGGACATCAGCATGCTTCTGCTTTTGATCAGGCTGCCAGATCTAGAGGA AATGGCAGCACAGCACTGCGGCCAGGATCTCAGCAGGAAGCTACAGAAGTGAGACCTG AACAGAAAATGCCTACACTGCTGAGAGTGTATATTGATGGACCACATGGAATGGGAAAAACAAC CACAACCCAGCTGCTGGTGGCTCTCGGATCTAGAGATGATATTGTGTATGTGCCTGAACCTATG ACATATTGGAGAGTGCTGGGAGCTTCTGAAACAATTGCTAATATCTATACAACACAGCATAGAC AAGGAGAAATTTCTGCCGGAGATGCTGCCGTGGTGATGACATCTGCTCAGATTACAAT GGGAATGCCTTATGCTGTGACAGATGCTGTGCTGGCACCACATATTGGAGGCGAAGCTGGAAGC TCTCATGCACCACCACCAGCACTGACACTGATTTTTGATCGGCATCCAATTTTCGCACTGCTGT GTTATCCGGCAGCAAGATATCTGATGGGAAGCATGACACCACAAGCCGTGCTGGCTTTTGTGGC TCTGATTCCACCAACACTGCCTGGAACAAACATCGTGCTGGGAGCTCTGCCTGAAGATAGACAT ATCGATCGGCTGGCCAAACGGCAGAGACCTGGAGAACGGCTGGATCTGGCCATGCTGGCTGCCA TTCGGAGAGTGTATGGCCTGCTGGCTAACACAGTGAGATATCTGCAGTGTGGAGGCTCTTGGAG AGAGGATTGGGGACAGCTGTCTGGCACAGCTGTGCCACCACAGGGAGCCGAACCACAGAGCAAT GCTGGACCACGACCACATATCGGAGACACACTGTTTACACTGTTTCGGGCACCAGAACTGCTGG CACCAAATGGAGACCTGTACAACGTGTTTGCCTGGGCTCTGGATGTGCTGGCTAAACGGCTGAG ATCTATGCATGTGTTTATCCTGGACTATGATCAGTCACCGGCCGGATGTCGCGATGCCCTGCTG CAGCTGACATCTGGGATGGTGCAGACACATGTGACAACACCTGGATCTATCCCAACAATCTGTG ATCTGGCTAGAACATTCGCTAGGGAGATGGGAGAGGCCAACTAATGAGGATCCCTCGAGAAGCT TGTCA SEQ ID NO: 22 HSV-TK dmNLS Al68H, CO & SC dmNLS = double mutated Nuclear Localization Sequence CO = codon optimized SC = splice corrected at 327 and 555 Kozak Sequence, Underlined gtcaGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGC CTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCaCTGCGgCCaGGATCT GAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG ACGGaCCaCACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA CGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC GCCAACATCTACACCACCCAGCACCGCCTGGACCAaGGCGAGATCAGCGCCGGCGACGCCGCCG TGGTGATGACCAGCGCCCAGATtACaATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGC aCCaCACATCGGCGGCGAGGCCGGCAGCAGCCACGCaCCaCCaCCaGCaCTGACCCTGATCTTC CACCCaATCGCaCACCTGCTGTGCTACCCgGCaGCaCGCTACCTGATGGGCtccATGA CaCCaCAaGCCGTGCTGGCCTTCGTGGCCCTGATCCCaCCaACaCTGCCCGGCACCAACATCGT GCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAG CGCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGC GCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCC aCCaCAGGGCGCCGAGCCaCAGAGCAACGCCGGaCCaCGaCCaCACATCGGCGACACCCTGTTC ACCCTGTTCCGgGCaCCaGAGCTGCTGGCaCCaAACGGCGACCTGTACAACGTGTTCGCCTGGG CCCTGGACGTGCTGGCCAAGCGCCTGCGCtccATGCACGTGTTCATCCTGGACTACGACCAGtc aCCgGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACa ACaCCCGGCAGCATCCCaACaATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGG CCAACTAATAGGGATCCCTCGAGAAGCTTgtca SEQ ID NO: 23 — MAP Kinase Kinase Nuclear Export Polynucleotide Sequence -H0- 2014/029814 C T GCAGAAAAAGCTGGAAGAGCTGGAACTGGATGGC SEQ ID NO: 24 MAP Kinase Kinase Nuclear Export Polypeptide Sequence T.QKKT. HT. *iT .DG

Claims

1. A polynucleotide encoding a mutated form of thymidine kinase from a human herpes simplex virus type 1 (HSV1-TK) and at least one pro-apoptotic agent, wherein the encoded HSV1-TK comprises at least two mutated amino acids in a nuclear localization sequence (NLS) at positions corresponding to amino acid residue 25, 26, 32, or 33 of SEQ ID NO:2, and wherein the mutated HSV1-TK increases cell kill activity relative to a wild-type HSV1-TK.

2. The polynucleotide according to claim 1, wherein the at least two mutated amino acids in the NLS comprises amino acid es at positions 32 and 33 of SEQ ID NO:2, wherein each amino acid residue is independently mutated to an amino acid chosen from the group consisting of glycine, serine, cysteine, glutamic acid, and aspartic acid.

3. The polynucleotide according to claim 1, wherein the at least two mutated amino acids in the NLS comprises amino acid residues at positions 25 and 26 of SEQ ID NO:2, wherein each amino residue is independently mutated to an amino acid chosen from the group consisting of: glycine, serine, cysteine, glutamic acid, and aspartic acid.

4. The polynucleotide according to claim 1, wherein the encoded mutated HSV1-TK further comprises a mutation at amino acid residue positions 167 or 168 of SEQ ID NO:2, or a combination thereof, to a polar, non-polar, acidic, or basic amino acid.

5. The polynucleotide according to claim 1, wherein the encoded d HSV1-TK further ses a mutation at amino acid residue on 167 of SEQ ID NO:2, to a polar, nonpolar , basic, or acidic amino acid.

6. The polynucleotide according to claim 1, wherein the encoded mutated K further ses a on at amino acid residue position 167 of SEQ ID NO: 2, to an amino acid ed from the group consisting of: histidine, lysine, cysteine, serine, and phenylalanine.

7. The cleotide according to claim 1, wherein the encoded mutated HSV1-TK r comprises a mutation at amino acid residue position 168 of SEQ ID NO:2, to a polar, nonpolar , basic, or acidic amino acid.

8. The polynucleotide according to claim 1, wherein the encoded mutated HSV1-TK further comprises a mutation at amino acid residue position 168 of SEQ ID NO:2, to an amino acid ed from the group consisting of: histidine, lysine, cysteine, serine, and phenylalanine.

9. The polynucleotide according to claim 1, wherein the encoded mutated K further comprises a nuclear export signal sequence.

10. The polynucleotide according to claim 9, n the nuclear export signal sequence is LQKKLEELELDG (SEQ ID NO: 24).

11. The polynucleotide according to claim 1, n the at least one optotic agent is a cancer suppressor.

12. The polynucleotide according to claim 11, wherein the cancer suppressor is p53 or Rb.

13. The polynucleotide according to claim 1, wherein the at least one pro-apoptotic agent is selected from the group consisting of p15, p16, and p21/WAF-1.

14. The polynucleotide according to claim 1, wherein the at least one optotic agent is selected from the group consisting of Bax, Bad, Bik, Bak, Bim, cytochrome C, apoptosisinducing factor (AIF), Puma, CT 10-regulated kinase (CRK), Bok, glyceraldehydephosphate dehydrogenase, Prostate Apoptosis Response Protein-4 (Par-4), Smac, Kinase C6, Fas, inhibitory PAS domain protein (IPAS), and Hrk.

15. A retroviral vector comprising the polynucleotide of claim 1 encoding the mutated modified HSV1-TK and the at least one pro-apoptotic agent.

16. The retroviral vector of claim 15, further sing a polynucleotide encoding for a PiT-2 or PiT-1 polypeptide.

17. The retroviral vector of claim 15, further sing a polynucleotide encoding for a targeting ptide.

18. The retroviral vector of claim 17, wherein the targeting polypeptide binds to an extracellular protein.

19. Use of a retroviral vector of claim 15 in the manufacture of a medicament for treating cancer in a patient in need thereof, wherein the retroviral vector is formulated to be administered to the t prior to the patient being administered a nucleoside analogue or a prodrug thereof.

20. The use of claim 19, wherein the iral vector is formulated to be administered intravenously, uscularly, subcutaneously, intra-arterially, intra-hepatic arterially, intrathecally , intra-peritoneally and/or intra-tumorally.

21. The use of claim 19, n a cell of interest is uced with the retroviral vector in vitro, and is formulated to be administered to the patient in need thereof.

22. The use of claim 19, wherein at least 1 x 109 TVP of the retroviral vector is formulated to be administered cumulatively to the patient in need thereof.

23. The use of claim 19, wherein the prodrug is administered between about 1-2 days after administration of the retroviral vector.

24. The use of claim 19, wherein the nucleoside analogue is ganciclovir.

25. Use of retroviral vector of claim 15 in the manufacture of a medicament for inducing cell kill activity in a cancer cell of interest, wherein: a. the cancer cell is ted with the medicament; b. the cancer cell is transduced with the polynucleotide encoding the modified HSV1-TK; c. the cancer cell is contacted with a nucleoside ue or a prodrug thereof, thereby inducing cell kill activity in vivo.

26. The use of claim 25, wherein the nucleoside analogue is ganciclovir.

27. The use of claim 25, wherein the cancer cell of interest is transduced in vivo in a t in need thereof.

28. The use of claim 25, n the cancer cell in step (b) is transduced in vitro, and subsequently administered to a patient in need thereof.