CA2143308A1

CA2143308A1 - Translocation signal facilitated nuclear delivery of macromolecules

Info

Publication number: CA2143308A1
Application number: CA002143308A
Authority: CA
Inventors: Thomas R. Barnett; Rathindra C. Das
Original assignee: Individual
Current assignee: Bayer Corp
Priority date: 1992-08-25
Filing date: 1993-08-24
Publication date: 1994-03-03
Also published as: IL106760A; KR950703650A; JPH08504565A; ZA936189B; FI950866L; IL106760A0; FI950866A0; AU674026B2; WO1994004696A1; EP0658210A1; NZ255870A; AU5088593A

Abstract

Methods for introducing proteins or nucleotide sequences into the nucleus using a novel receptor-mediated delivery sys-tem. The delivery system construct includes a cell receptor-binding domain, a cytoplasmic translocation domain, and a nuclear translocation signal domain. This system can transport functional macromolecule that will act once internalized into the nucleus.

Description

~143308 W O 94/04696 PC~r/US93/0794 TITLE: TRANSLOCATION SIGNAL FACILITATED NUCLEAR
DELIVERY OF MACROMOLECULES

FIELD OF THE INVENTION
S The invention relates to methods of introducing foreign materials into a cell nucleus. More particularly, the present specification discloses methods for transporting nucleotide sequences or proteins into the nucleus using a novel translocation signal facilitated delivery system.
BACKGROUND
Individuals suffering from genetic diseases manifested by a deficiency in the expression of certain necessary proteins require augmentation with therapeutic doses of such proteins to lead normal lives. Currently, such treatment is mainly that of lifetime maintenance w.ith periodic, exogenous introduction of the required protein. Such periodic treatment is cumbersome, expensive, and sometimes hazardous (such as for hemophiliacs who have a relatively high exposure rate to HIV) .
It is desirable to utilize biomolecular manipulation to augment such protein deficiencies by introducing DNA (which code for the deficient proteins), and other nucleotide sequences and polypeptides (which regulate the expression of the deficient proteins) into the cells of patients suffering from gene~ic deficiencies. For example, such treatment involves the introduction of genes and regulators for factor VIII for hemophiliacs and al- antitrypsin for patients suffering from hereditary emphysema or adult respiratory distress syndrome (ARDS).
Such approaches can even be extended to re-transform aberrant tumor cells in cancer patients. The most attractive manner for achieving such therapeutic transformation is to deliver a genc coding for the deficien gene product into the nucleus of somatic cells. In vitro delivery of foreign DNA into mammalian cells for gene expression has been achieved by three distinct approaches. The firsl approach takes advantage of the natural ability of viruses to infect cells and express 3 5 viral DNA in thc form of specific RNA and protein species (Cournoyer et al., 1991, "Gene transfer of adenosine de~min~ce into primitive human hematopoetic progenitor cells", Human Gene Therapy, 2:203; Rosenberg et al., 1990, "Gene transfer into humans: immunotherapy of patients with advanced melanoma using infiltrating Iymphocytes modified by ~,~433~

WO 94/04696 PCr/US93/07945 retroviral gene transduction", New England Journal of Medicine, 323:570). In particular, advantage has been taken of m~mm~lian retroviruses as vector systen1s that permit infection of a variety of cell types and allow for expression of many different foreign genes.
5 Retroviruses and their recombinant forms are thought to bind to cells via specific receptors on the cell surface, after which they are internalized by endocytosis. Once endocytosed, the virus is able to evade the endosome-lysosome pathway by a mechanism which is thought to disrupt the endosome, escape degradation and permit entry into the cell 10 nucleus.
A second approach fuses artificial lipophilic vesicles containing exogenous DNA with a cellular target (Felgner et al., 1987, "Lipofection:
a highly efficient, lipid-mediated DNA- transfection procedure", Proc.
Natl. Acad. Sci. USA, 84:7413). Delivery of DNA to the nucleus via 15 lipophilic vesicle fusion is thought to be possible because it is hypothesized that endosome- associated degradation might be bypassed.
A third, non-specific approach for introduction of foreign DNA
into cells is achieved by mixing exogenous DNA with a polycationic support, such as DEAE-dextran (McCutchan et al., 1968, "Enhancement of 20 the infectivity of simian virus 40 deoxyribonucleic acid with diethyl aminoethyl-dextran", J. Natl. Cancer Inst., 41:351); or by complexing with calcium phosphate (Graham et al., 1973, "A new technique for the assay of infectivity of human adenovirus 5 DNA", Virology, 52:456).
The exogenous DNA mixture with polycationic support or calcium 25 phosphate is then incubated with live cells (i.e. a transfection step).
Uptake or endocytosis of DNA can be monitored by subsequent selection of the expressed phenotype, typically by complementation or by antibody-reactive surface markers. The mechanism of DNA uptake by the cells is largely unknown, but it is generally accepted that the DEAE-30 dextran or calcium phospha~e protects the DNA from the nucleaseactivity of Iysosomal cell compartments, followed by escape to the nucleus where expression occurs. Increased efficiency of expression using the transfection method can be achieved when Iysosomotropic agents, such as chloroquine (Luthman et al., 1983, "High efficiency 35 polyoma DNA transfection of chloroquine treated cells", Nucl. Acids Res., 11: 1295), are included in the transfection mixtures. Lysosomotropic agents apparently reduce Iysosomal destruction of DNA by increasing the relatively low pH necessary for activation of degradation.

3 n s ~
WO 94/04696 PCr/US93/07945 Thus, several in vitro methods have been proposed for introduction of foreign DNA into mammalian cells. However, these known methods have inherent drawbacks in use. In order to translate the in vitro approaches of gene expression to the problem of delivering ,. S genetic material to the nucleus to express therapeutic amounts of gene product, many problems and considerations have to be addressed.
These problems include, but are not limited to the practical administration of a gene to an individual suffering from a particular disease amenable to therapy; targeting of the gene of interest to a particular cell-~ype or organ; efficient uptake of the gene by the cells;
-- targeting of the gene to the nucleus; and efficient and sustained expression of the gene product. For example, while valuable as in vitro tools, retroviruses have considerable problems when used in vivo, including a very broad cell type specificity, the requirement for dividing cells to permit replication of the genome, inefficient expression of an inserted gene once in the nucleus, and questions of human safety.
~fforts have been made in the use of specific cell-surface receptors to mediate uptake of protein that is electrostatically- coupled to a piece of DNA that is capable of expressing a gene product of interest. This was first made possible by the observation that a complex consisting of orosomucoid-coupled poly-L-lysine could bind by salt bridges a plasmid DNA that coded for the bacterial-derived chloramphenicol acetyltransferase (CAT) gene. Presentation of this complex to cells permitted uptake by asialoglycoprotein receptors and short term expression of the CAT gene (Wu et al.. 1989, "~vidence for targeted gene delivery to HepG2 hepatoma cells in vitro", Biochemistry, 27:887). However, the number of cells actually expressing the gene product of interest was several orders of magnitude lower than the 30 number endocytosing the complex. suggesting considerable intra-cytoplasmic degradation of the complex after uptake.
Another approach was provided by the observation that after infection of a host cell, adenovirus. Iike other viruses, evades intracellular destruction and targets its genome to the cell nucleus 35 (Curiel et al., 1991, '-Adenovirus enhancement of, transferrin-polylysine-mediated gene delivery", Proc. Natl. Acad. Sci. USA, 88:8850).
Advantage was taken of the ability of the adenovirus particle to "uncoat'` its capsid proteins and permit early escape from the endosome-lysosome pathway. This group added adenovirus particles to a 2~4~Q~
W O 94/04696 PC~r/US93/07945 mix of transferrin-poly-L-lysine that was electrostatically linked to DNA in the range of a few kilobases to nearly fifty kilobases to mediate enhanced expression of, in this case, the product of thc luciferase gene (Cotten et al., 1992, "High-efficiency receptor-mediated delivery of 5 small and large (48 kilobase) gene constructs using the endosome-disruption activity of defective or chemically inactivated adenovirus particles", Proc. Natl. Acad. Sci. USA, 89:6094). Without added adenovirus capsids, expression of the luciferase gene was quite low, while with the adenovirus capsid, expression increased several orders of magnitude 10 (Wagner et al., 1992, "Coupling of adenovirus to transferrin-polylysine/DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes", Proc. Natl. Acad. Sci. USA, 89:6099). However, even with viral capsid uncoating, transport of transferrin-DNA complexes to the nucleus for expression is essentially 15 random and introduces an element of uncertainty to such procedures.
A major disadvantage of the adenovirus-facilitated gene delivery technique is that success relies on the tedious preparation of chemically-derived protein-DNA-virus complexes of unknown quality to achieve the enhanced levels of expression reported. A funher 20 drawback is the requirement of the concomitant expression of both transferrin and adenovirus receptors on the desired cell targets.

SUMMARY OF THE INVENTION
The present invention is a translocation signal facilitated system 25 for providing efficient, reproducible and targeted delivery of protein or DNA-protein complexes with therapeutic value, to the nuclei of mamm~lian cells. our approach takes advantage of the natural ability of some proteins, not just viruses, to enter cells and perform specific functions, such as directing their way out of endosomes into cytoplasm 30 with the help of a cytoplasmic trans- location signal, and further directing their way to the nucleus with the help of a specific nuclear targeting signal.
The present disclosure demonstrates cell specific targeting and intracellular translocation with exotoxin A of Pseudomonas aeruginosa 35 which generally infects patients at the site of surface injury, but which can also target its virulence at fibroblast cells and systemically, primarily to the liver, and secondarily to organs such as the kidney and spleen. ~xotoxin A is composed of four domains. which are organized starting from the amino terminus as domains la. Il, Ib, and Ill (Allured 3 3 0 8;
~0 94/04696 PCI/US93/07945 et al., 1986, "Structure of exotoxin A of Pseudomonas aeruginosa at 3.0-Angstrom resolution". Proc. Natl. Acad. Sci. USA, 83:1320) (Figure 1).
Domain la (amino acids 1-252) binds the exotoxin specifically to a cell-surface receptor; domain Il (amino acids 253-364) is the region of 5 the exotoxin that is specifically cleaved after activation in the low pH
compartment of the endosome and translocates the distal regions of the exotoxin out of the endosome; domain III (amino acids 405-613) contains the ADP- ribosylating activity of the toxin that inactivates ribosome associated protein elongation factor 2; domain Ib appears to be a structural~ rather than functional, domain (amino acids 365-404). The features of the exotoxin protein which are important for each step in its uptake and delivery to the cell cytoplasm are described in Siegall et al., l9X9, "Functional analysis of domains II, Ib and III of Pseudomonas exotoxin", J. Biol. Chem., 264: 14256.
In addition. chimeric molecules can be constructed which allow attachment of other non-toxin-related receptor-binding domains to the ADP-ribosylation domain III for targeting toxin activity to the cytoplasm of certain cells for the purpose of cancer chemotherapy. It has also been demonstrated that exotoxin A domains Ia and II can be 20 used to deliver an unrelated domain, in this case, a bacterial nuclease, barnase, to the cytoplasm of rn~mmali~n cells (Prior et al.. 1992, "Translocation mecli~t~d by domain Il of Pseudontonas exotoxin A:
transport of barnase into the cytosol", Biochemistry, 31 :3555).
We describe a novel and efficient system that overcomes the 25 inadequacies of other systems for delivering both genes and proteins of therapeutic use to the nuclei of cells. A schematic representation of the molecular system designed for the nuclear targeting and delivery of DNA or proteins is depicted in Figure 2. This figure shows the preferered configuration of the critical domains needed f or targeting a 30 macromolecule to the nucleus for therapeutic intervention.
The critical features of the present delivery system are "X" the receptor-binding domain; "II", the cytoplasmic translocation domain;
"NTS", the nuclear translocation signal domain; and " Z", the functional macromolecule that will act once internalized into the nucleus. By way 35 of example, Figure 3 shows the construction of a specific carrier protein and Figure 4 shows a schematic representation of the protein carrier-mediated delivery of a target protein to a cellular receptor for the exotoxin A binding domain. In the embodiment depicted in Figure 3, the receptor-binding domain ("X") is domain la derived from the 2~ ~33~ ~
WO 94/04696 PCl/US93/07945 Pseudomonas exotoxin A gene, and the functional domain (" Z") is B-galactosidase, the nuclear delivery of which is measured by the development of blue color in the presence of 5-bromo-4-chloro-3-indoyl-B-D- galactopyranoside (X-gal) substrate. Starting from the amino terminus of the construc~, the delivery system consists of domains la and Il from Pseudon~onas exotoxin A, a nuclear targeting signal (NTS) from SV40 T-antigen, and a functional version of 0-galactosidase. Domain Ia provides a natural means for directing the chimera to a particular cell type containing exotoxin A receptors such as fibroblasts, or to an organ such as the liver, by binding to a specific cell-surface receptor.
The protein-carrier complex is internalized into the cell by endocytosis, and the complex becomes engulfed in endosomes. During the course of its maturation, the pH of the endosome becomes more acidic, whereupon a pH-driven cleavage of domain II occurs, and it is the activation of this domain that is the key to the translocation of the distal portion of the protein (part of domain II, NTS, and B-galactosidase) to the cytoplasm (see Figure 3). Once in the cytoplasm, the presence of a nuclear targeting signal (NTS), in this case derived from a viral protein of SV40 that is normally translated in the cytoplasm but functions in the nucleus, interacts with a cytoplasmic protein that promotes rapid translocation of the protein-carrier complex to the nucleus. Once in the nucleus, the polypeptide domain, in this case derived from B-galactosidase, is free to function appropriately.
It will be appreciated by those skilled In the art that domain la could be substituted by other receptor-binding domains (for example, transforming growth factor alpha (TGF-aL); or other toxin- derived ligands such as from diphtheria toxin) and that B-galactosidase can be equally substituted by other protein domains, for example, transcription 3 0 factors. In the latter case, it will be appreciated that when other proteindomains that may naturally be found in the nucleus are substituted for B-galactosidase, they may already have a nuclear translocation signal (NTS) coded within them, so that inclusion of additional NTS may be unnecessary. Similarly, one skilled in the art can appreciate that the 3 5 SV40 nuclear translocation signal domain can be substituted with others such as yeast alpha-2, GAL 4, etc.
It will also be appreciated tha~ neither the first nor the las~
protein domains need be contiguous polypeptide chains, but rather could be synthesized independently and attached lo the amino- or 2 1 4 ~ ~ 0 8 94/04696 PCI`/US93/07945 carboxyl- termini by chemical modification. It will be further appreciated that for the purpose of delivering a protein domain to the nucleus, non-protein receptor-binding domains could be substituted for domain Ia in the delivery system described above. Finally, in one 5 embodiment the delivery of a chimeric molecule, such as above-described, can be targeted in vivo simply by injection into the systemic blood circulation.
For the purpose of delivering a nucleic acid, such as DNA, to the nucleus, the only modification of the chimeric construct described in 10 Figure 2 is that domain " Z" is no longer a functional polypeptide domain, but rather a DNA-binding domain. This DNA- binding domain is attached by electrostatic binding either specifically or non-specifically to a piece of DNA, comprising either a sense or antisense oligonucleotide, or an expressible gene that includes replication, 15 regulatory, transcriptional and/or translational sequence signals.
Figure S shows a pictorial representation of a protein carrier designed to deliver nucleic acid (DNA) to a cell nucleus for gene therapy. Figure 6 shows the construction of a protein carrier to be used for DNA binding, in which the receptor-binding domain ("X" ) is 20 domain la derived from the Pseudomonas exotoxin A gene, domain II and the NTS domains are as described above, and domain "Z" is a stretch of poly-L-lysine which is used for electrostatic interaction with a plasmid DNA molecule that codes for galactosidase.
As described previously. domain la is used for binding of the 25 delivery system-DNA complex molecule to a cellular receptor. After endocytosis and internalization, and maturation of the endosome, pH
dependent cleavage of domain Il occurs and translocation of the truncated complex to the cytoplasm ensues, where the NTS domains and their respective binding proteins transport the complex into the 30 nucleus. The NTS domain binds to a cytoplasmic protein which mediates translocation of the complex to the nucleus. Once translocated, nuclear processes can unwind the DNA-binding domain from the DNA itself, followed by transcription and translation of RNAs coding for therapeutic proteins; if appropriate, replication of the targeted DNA
35 may ensue.
It is understood that there are many other different examples of polypeptide domains that bind DNA including poly-L-lysine and poly-D-lysine, repeats of nuclear translocation signal sequences ("poly-NTS"), ornithine, putrescine, spermidine. spermine. histones and other non-?

WO 94/04696 7 1 4 3 3 ~ 8 PCI/US93/07945 sequence-specific basic DNA-binding proteins~ and sequence-specific DNA-binding proteins like homeobox domains. Those skilled in the art will appreciate that other polycationic macromolecules can be substituted as means for connecting DNA to the delivery system such as 5 synthetic chemical linkers. With the exception of poly-L- (or -D-) Iysine, other types of DNA-binding proteins often have NTS signals coded within them. In such cases, there would be no need for additional NTS domains in the final constructs. These DNA-binding domains can be contiguously translated as part of the chimeric molecule or attached to 10 the basic construct by chemical modification. An alternative to the use - of poly-L-lysine is to include within a DNA construct targeted for nuclear expression a short specific DNA sequence that could be bound by a specific DNA-binding protein, for example, a homeobox domain or other transcription factor. Free poly-L-lysine or poly-D-lysine can then 15 be added to aid in the collapse of the DNA molecule itself to facilitate subsequent gene expression. As above, it will be recognized that the receptor-binding domain Ia of Pseudor~lonas exotoxin A can be substituted with other receptor-binding domains.
As the following Detailed Description outlines, the key to 20 successful delivery of DNA or proteins to the nucleus pursuant to the present invention include (1) the use of Pseudomonas exotoxin A
domain II or its functional equivalent to mediate translocation to the cytoplasm, and (2) the use of a nuclear targeting signal to mediate translocation to the nucleus.
DETAILED DESCRIPTION OF THE INVENTION

Cloning of Pseudo~lonos exotoxin A (ETA) ~ene 3 0 Genomic DNA is prepared according to the method described by Marmur et al., 1961, "A procedure for the isolation of deoxy-ribonucleic acid from micro-organisms". Journal of Molecular Biology~ 3:208, from bacterial strain Pscudomonas aeruginosa PA103 (American Type Culture Collection 29260), originally described as producing exotoxin A (Liu, P., 1966, "The roles of various fractions of Pseudomonas aeruginosa in its pathogenesis. III. Identity of the lethal toxins produced in vitro and in vivo", Journal of Infectious Diseases, 116:481).
Genomic DNA is cleaved wi~h restriction endonucleases NotI and ~coRI, then is electrophoretically separated on a 0.8% low- melting ~ ~ 4~ ~ ~8 WO 94/04696 ~ =` PCT/US93/07945 agarose gel, from which a region of ~2 to 2.6 kilobase pairs is excised (the expected size of the exotoxin A gene segment is 2.3 kb, Gray et al., 1984, "Cloning, nucleotide sequence, and expression in Escherichia coli of the exotoxin A structural gene of Ps~udomollas aeruginosa", Proc.
Na~l. Acad. Sci. USA., 81 :2645) and the DNA eluted. Purified NotI-EcoRI
DNA is ligated to the phosphatased arms of bacteriophage ; ~gt 11 previously cleaved with Notl and EcoRI (Promega Corp., Madison, WI) in a reaction using T4 DNA ligase f or 16 hours at 15C.
DNA is packaged into infectious panicles using complementing 10 packaging extracts obtained from Stratagene Corp. (San Diego, CA).
Approximately 1000 recombinant ~ particles are plated on indicator Esclleric~tia coli Y1090 and lifted onto nitrocellulose filters. DNA is Iysed in situ as described in Maniatis et al., 1982, Molecular Cloning, A
Laboraton~ Manual. Cold Spring Harbor, NY. Filters are then screened 15 by hybridization with a DNA probe corresponding to the Ia domain of Pseudomonas exotoxin A (ETA), prepared by PCR amplification of cleaved Pseudomonas aeruginosa PA103 DNA using oligonucleotides GTlOSF
(S '-GGATCCTCATGAGCGCCGAGGAAGCCTTCGACCTC) (SEQ. lD. NO. I ) and 20 GT103R (5'-AAGCTTGGGAAAGTGCAGGCGATGACTGAT) (SEQ. ID. NO.2) in the presence of digoxigenin-dUTP (Boehringer Mannheim Biochemicals, Indianapolis~ IN).
Positive plaques are amplified for small-scale preparation of DNA
which is recloned for easy manipulation into the Bluescript KS+ plasmid 25 vector (Stratagene CorpSan Diego. CA). The 2.3 kilobase ETA genomic DNA is used as a source of template f or further amplifications and modifications of different ETA domains.

30 Cloning of Pseudor7l0nas ~TA domains la and II

The 2.3 kb DNA from Example I is used for polymerase chain reaction-mediated amplification of the binding (Ia) and cytoplasmic ., translocation (II) domains of the ETA gene. oligonucleotides GT105F
35 (5 '- GGATCCTCATGAGCGCCGAGGAAGCCTTCGACCTC) (SEQ. ID. NO. l) and GT106R2 (5`-A~GCTTGGTGCCCTGCCGGACGAAGCGCT~ (SEQ. ID. NO.3) are used to amplify and for cloning DNA sequences coding for domains la and II, followed by downstream expression of the domains as non-secreted polypeptides in bactcrial and insect cells.

WO 94/04696 2 1 1 3 3 ~ 8 PCI'/US93/07945 Incorporated into the 5' ends of the oligonucleotides are restriction sites, BamHI and HindIII (underlined above) to facilitate downstream cloning and manipulation of domains. To achieve secretion of polypeptides having N-terminal domains la and II in the bacterial expression plasmid, pSE380 (Invitrogen Corporation, San Diego, CA), a second version of the clone is prepared using a forward oligonucleotide that primes within the DNA sequence coding for the Pseudomonas exotoxin A signal peptide sequence (GTOOlSPiF; TCATGATCCTGATACCCCATTGGATTCCCCTG) (SEQ. ID. NO. 4); for secretion in the insect cell expression system, a forward oligonucleotide coding within the signal sequence of an abundant baculovirus envelope glycoprotein, gp67 (Stewart et al.,. 1991, "Construction of an improved baculovirus insecticide containing an insect-specific toxin gene", Nature, 352:85) is used (GTO02SPF;

AAGATGGTAAGC~GCTAl l~-l l l'l'ATATGTC;(_l'l'l l~l~Tl~GC~
GCGGAGGAAGCCTTCGACCTC) (SEQ. ID. NO. 5). Figure 7a shows the modifled forms of domains Ia and II as a BamHI-BspHI/HindIII DNA cacsett~.

Prevaration of Nuclear Translocation Signal In order to facilitate targeting of proteins or protein-DNA
complexes to the nucleus, a well-characterized nuclear translocation signal (NTS) domain from the mammalian virus, SV40 (Garcia-Bustos el al., 1991, "Nuclear protein localization" Biochemica et Biophysica Acta, 1071:83) is included in the preparation of constructs.
Two overlapping synthetic oligonucleotides with complementary 5' HindIII restriction sites are used to prepare the SV40 NTS domain, namely, GT108F; 5'-AGCTTCCTAAGAAGAAACGTAAGGTCA (SEQ. ID. NO. 6), and GT108R; 5'-AGCTTGAC(~I~ACGTTT~,'TTCTTAGGA (SEQ. ID. NO. 7). Figure 7b shows amplifed NTS domain produced by this method as a HindIII-HindIII cassette. This cassette is then ligated by T4 DNA ligase at the 3' end of ETA domains la and II. The position of the Hindlll-Hind III SV40 3 5 NTS cassette relative to ETA IA-II domain cassette is shown in Figure 7d.

Cloning of ~.~c)lcricllia coli lac z ~ene 1 ~

~ ~ ~ 3 3 ~ 8 WO 94/04696 PCI`/US93/0794~

As a means of visually detecting proteins that are targeted to the nucleus, constructs are prepared that include an amino-terminal truncated version of the bacterial lac Z ('lac Z) gene coding for ~-galactosidase. This DNA segment was obtained by amplification of the lac 5 Z gene-containing plasmid, pCHl lO (Pharmacia, Piscataway, NJ), using oligonucleotides GT107F
(5'- AAGCTTCAACGTCGTGACTGGGAAAACCCTGGCGTT) (SEQ- ID. NO.8) and GT107R (5- CTGCAGCTATTAl l l l l~iACACCAGACCAACTGGTAATG) (SEQ. ID. NO.
9). Figure 7c shows the 'lac Z gene sequence as a HindIII-PstI cassette.

Con.struction of a Protein Carrier for Nuclear Delivery of a Target Protein: FTA domains la and Il/ NTS / ei-s~alactosidase Figure 7d shows the assembly of DNA cassettes coding for Pseudontonas ETA domains la and II, the SV40 NTS domain, and the 'lac Z
gene. The ETA IA-II DNA cassette is ligated to the NTS and lac Z DNA
cassettes by T4 DNA ligase using the common HindIII restriction sites that are included within the PCR amplification primers. The final DNA
20 construct is inserted into plasmid vectors for expression in the baculovirus system using onc of scveral baculovirus expression plasmids (for example, PVL1393; Webb et al., 1990, "Expression of proteins using recombinant baculoviruses", Technique - A Journal of Methods in Cell and Molecular Biology, 2:173), or by expression in a 25 bacterial host by incorporation into a high expression plasmid vector, like pET, which uses T7 promoter-specific expression (Studier et al., 1986, "Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned gene", J. Mol. Biol., 189:113).
Protein constructs expressed in either baculovirus or bacteria 30 are purified from cell Iysates and/or media over anti-~- galactosidase antibody columns linked via protein A. Proteins thus prepared are suitable for targeting for targeting functional ~- galactosidase to the nucleus by in vitro incubation with exotoxin A-sensitive mammalian cells (for example, L-M cells, American Type Culture Collection CCL 1.2 35 or Chang liver cells, American Type Culture Collection CCL 13) and/or after injection into the mammalian blood circulation for in vivo targeting to eithcr the liver and/or secondary organ sites.
- Successful nuclear targeting is assaycd histochemically by the conversion of the colorless X-gal substrate to blue~ indicating functional 1 ~

21~3~8 ~
WO 94/04696 - ` ~ PCI/US93/07945 B-galactosidase. Similar constructs can be prepared for use with other domains substituted for ~-galactosidase and/or ETA Ia domains.

S Co~truction of FTA domains Ia and Il/ NTS / cationic polypeptide Figure 8 shows the preferred configuration for a continuous polypeptide that has a polycation stretch at the "Z" domain. In this example, we use poly-L-lysine as the polycation stretch. A polylysine 10 polypeptide segment is generated from a synthetic DNA segment of ~200 - 300 bases of poly AAA/AAG (Iysine codons) containing an HindIII
restriction site at its 5' end and a Pstl site at its 3' end (as in 'lac Z). Just proximal to the Pstl site is placed a stop codon like TAG, TGA or TAA to terminate translation.
As an alternative to the use of polylysine, a DNA segment containing as many as 20-30 NTS repeats derived from the SV40 sequence (since the SV40 nuclear targeting signal is polycationic), or a smaller number of repeats if they are derived from other longer nuclear targeting signals, can be used (Figure 9). Any one of these 20 different polycationic segments can be ligated at the 3' end of the core construct consisting of ETA IA-II/ NTS (BamHI-HindIII-HindIII) cassette (refer to Figure 7d).
As a further alternative to incorporating polycation substitutions for the l~-galactosidase domain as continuously translated polypeptides, 25 it will be appreciated that polylysine, for example, can be covalently coupled by chemical modification to a bacterial or baculovirus -expressed ETA IA-II-NTS core construct in order to achieve a similar effect (Figure 10).

Construction of F.TA domains la and Il/ NTS / DNA binding protein Figure 11 shows an alternative method for preparing a DNA
binding protein domain (domain "Z"). This polypeptide domain is 3 5 derived from any of several nuclear proteins that bind specific DNA
sequences, for example, homeobox domains or yeast GAL4 protein. These domains are amplified by the PCR from genomic DNA or from cDNA
coding for these domains. Included at the 5' and 3' ends of the amplified domain are the HindIII and Pstl restriction sites that are also used for l--wo 94/04696 2 1 4 ~ 3 0 8 PCI/US93/07945 constructing 'lac Z or polylysine domains. In our example, a shorl defined DNA sequence is included in the construction of a DNA gene that will be targeted for expression in the nucleus. one or more of these domains can be used to bind DNA; subsequent complex formation would include addition of non-covalently added basic proteins or polycations, like poly-L-lysine (or poly-D-lysine).

Construction of a Protein Carrier-DNA Complex for Nuclear Delivery of DNA for ~cpres~ion: F.TA domains la ~nd Il/ NTS / poly-L-lysine linked to a plasmid coding for B-~alactosidase Figure 12 shows the preferred method for preparing a protein carrier that can deliver a DNA construct to the nucleus of a mammalian cell for the purpose of expressing a gene coding for ~- galactosidase.
The ETA IA-II domains described above are ligated to the NTS domain by T4 DNA ligase using the common HindlII restriction sites that are included within the amplimers. This DNA construct is insened into plasmid vectors for expression in the baculovirus system using one of several baculovirus expression plasmids (for example, pVL1393; Webb et al., 1990, "Expression of proteins using recombinant baculoviruses", Technique - A Journal of Methods in Cell and Molecular Biology, 2:173), or by expression in a bacterial host by incorporation into a plasmid vector, Iike pET, which uses T7 promoter-specific expression (Studier et al., 1986, "Use of bacteriophage T7 RNA polymerase to direct selective high- level expression of cloned gene", J. Mol. Biol., 189:113).
Expressed protein carriers are purified from cell Iysates and/or media by conventional ion exchange chromatography over an anionic exchange column such as carboxymethyl-Sepharose, that will bind basic proteins. Commercially available poly-L-lysine (40, 000 mol. wt.) is then coupled to the ETA IA-II / NTS domain by using conventional NHS
(N-hydroxysuccinimide) chemistry. Uncomplexed poly-L-lysine is removed by chromatography over Sephadex G-100 (or its equivalent) in phosphate-buffered saline.
The resultant purified protein carrier is then incubated with pCHl 10, a commercially available m~mm~ n expression plasmid coding for l~-galactosidase (Pharmacia, Piscataway, NJ), in 2M NaCI, 10 mM
Tris-HCl, pH 7.5, I mM EDTA and diluted down to 150 mM NaCI by dialysis.
The formation of protein-DNA complexes is determined by gel 21~33~$. ~
W O 94/04696 ~ ' PC~r/US93/0794~

electrophoresis on 1% agarose, where plasmid DNA alone migrates in its supercoiled form at a relative molecular marker weight position of 7.2 kb, while complexes of supercoiled DNA with the ETA la-II/NTS/poly-L-lysine migrate at the top of the agarose gel, indicating very high 5 molecular weight complexes. Uncomplexed DNA is separated by size exclusion chromatography on Sepharose gel bead matrices. Protein carrier-DNA complexes prepared in this way are suitable for targeting to the nucleus by in vitro incubation with exotoxin A-sensitive m~mm~ n cells (for example, L-M cells, American Type Culture 10 Collection CCL 1.2 or Chang liver cells, American Type Culture Collection CCL 13) and!or after injection into the m~mm~ n blood circulation for in vivo targeting to liver and/or secondary organ sites.
Successful nuclear targeting can then be assayed histochemically by the conversion of the colorless X-gal substrate to blue, indicating 15 functional ~-galactosidase.

t ~

~0 94/04696 2 1 4 3 3 0 8 PCr/US93/07945 SEQUENCE LISTING
(I) GENERAL INFORMATION:
( i ) APPLICANT: Miles Inc.
(Thomas R. Barnett and Rathindra C. Das) (ii) TITLE OF INVENTION: TRANSLOCATION SIGNAL FACILITATED
NUCLEAR DELIVERY OF MACROMOLECULES
(iii) NUMBER OF SEQUENCES: 9 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Miles Inc.
(B) STREET: 400 Morgan Lane (C) CITY: West Haven (D) STATE: Connecticut (E) COUNTRY: USA
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(D) SOFTWARE: Word Perfect 5.1 (vi) CURRENT APPLICATION DATA:
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(C) CLASSIFICATION:
(vii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Barbara A. Shiemi, Esq.
(B) REGISTRATION NUMBER: 29,862 (C) REFERENCE/DOCKET NUMBER: MWH 314 (viii) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (203) 937-2340 (B) TELEFAX: (203) 937-2795 a) INFORMATION FOR SEQUENCE ID NO: I
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 nucleo~ides (B) TYPE: nucleic acids (C) STRANDEDNESS: single strand (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:
Other nucleic acid - oligonuleotide primer WO 94/04696 2 1 4 ~ 3 ~ ~ PCr/US93/0794~

(iii) PUBLICATION INFORMATION:
(A) AUTHORS: Gray et al.
(B) JOURNAL: Proc. Nall. Acad. Sci. USA
(C) VOLUME: 81 (D) PAGE: 2645 (E) DATE: 1984 (iv) SEQUENCE DESCRIPTION: SEQ ID NO: 1 (3) INFORMATION FOR SEQUENCE ID NO: 2 (i) SEQUENCECHARACTERISTICS:
(A) LENGTH: 30 nucleotides (B) TYPE: nueleic aeids (C) STRANDEDNESS: single strand (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:
Other nueleie aeid - oligonuleotide primer (iii) PUBLICATION INFORMATION:
(A) AUTHORS: Gray et al.
(B) JOURNAL: Proc. Natl. Aead. Sei. USA
(C) VOLUME: 81 (D) PAGE: 2645 (E) DATE: 1984 (iv) SEQUENCE DESCRIErrlON: SEQ ID NO: 2 (4) lNFORMATION FOR SEQUENCE ID NO: 3 (i) SEQUENCECHARACTERISTICS:
(A) LENGTH: 29 nueleotides (B) TYPE: nueleie aeids (C) STRANDEDNESS: single strand (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:
Other nueleic acid - oligonuleotide primer (iii) PUBLICATION INFORMATION:
(A) AUTHORS: Gray et al.
(B) JOURNAL: Proc. Natl. Aead. Sei. USA
(C) VOLUME: 81 (D) PAGE: 2645 (E) DATE: 1984 2143~0X
_ W O 94/04696 ` ! PC~r/US93/07945 (iv) SEQUENCE DESCRIPTION: SEQ ID NO: 3 (5) INFORMATION FOR SEQUENCE ID NO: 4 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 nucleotides (B) TYPE: nucleic acids (C) STRANDEDNESS: single strand (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:
Other nucleic acid - oligonuleotide primer (iii) PUBLICATION INFORMATION:
(A) AUTHORS: Gray et al.
(B) JOURNAL: Proc. Natl. Acad. Sci. USA
(C) VOLUME: 81 (D) PAGE: 2645 (E) DATE: 1984 (iv) SEQUENCE DESCRIPTION: SEQ ID NO: 4 T~ATGATCCT GATACCCCAT TGGATTCCCC TG 32 3 0 (6) INFORMATION FOR SEQUENCE ID NO: S
(i) SEQUENCECHARACTERISTICS:
(A) LENGTH: 138 nucleotides 3 5 (B) TYPE: nucleic acids (C) STRANDEDNESS: single strand (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:
Other nucleic acid - oligonuleotide primer (iii) PUBLICATION INFORMATION:
(A) AUTHORS: Stewart et al.
(B) JOURNAL: Naturc (C) VOLUME: 352 (D) PAGE: ~5 (E) DATE: 1991 (iv) SEQUENCE DESCRIPTION: SEQ ID NO: 5 ATAAGGAACA CACAAGCAAG ATGGTAAGCG CTAl l~l l l l 80 ATATGTGCTTl~ CGGCGCATTCTGC( l l l~CG 120 (7) INFORMATION FOR SEQUENCE ID NO: 6 1 ~

6 ~ 3 3 ~ 8 PCr/US93/07945 (i) SEQUENCECHARACTERISTICS:
(A) LENGTH: 27 nucleotides (B) TYPE: nucleic acids (C) STRANDEDNESS: single strand (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:
Other nucleic acid - oligonuleotide primer (iii) PUBLICATION INFORMATION:
(A) AUTHORS: Benditt et al.
(B) JOURNAL: Proc. Natl. Acad. Sci. USA
(C) VOLUME: 86 (D) PAGE: 9327 (E) DATE: 1989 (iv) SEQUENCE DESCRIPTION: SEQ ID NO: 6 (8) INFORMATION FOR SEQUENOE ID NO: 7 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 nucleotides (B) TYPE: nucleic acids (C) STRANDEDNESS: single strand (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:
Other nucleic acid - oligonuleotide primer (iii) PUBLICATION INFORMATION:
(A) AUTHORS: Benditt et al.
(B) JOURNAL: Proc. Natl. Acad. Sci. USA
(C) VOLUME: 86 (D) PAGE: 9327 (E) DATE: 1989 (iv) SEQUENCE DE~SCRIPTION: SEQ ID NO: 7 (9) INFORMATION FOR SEQUENCE ID NO: 8 (i) SEQUENOECHARACTERISTICS:
(A) LENGTH: 36 nucleotides (B) TYPE: nucleic acids (C) STRANDEDNESS: single strand (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:

WO 94/04696 2 1 ~ 3 ~ O ~ PCI/US93/07945 Other nucleic acid - oligonuleotide primer (iii) PUBLICATION INFORMATION:

(A) AUTHORS: Kalnins et al.
(B) JOURNAL: EMBO J.
(C) VOLUME: 2 (D) PAGE: 593 (E) DATE: 1983 tiV) SEQUENCE DESCRIPTION: SEQ ID NO: 8 (10) INFORMATION FOR SEQUENCE ID NO: 9 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 nucleotides (B) TYPE: nucleic acids (C) STRANDEDNESS: single strand (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:
Other nucleic acid - oligonuleotide primer (iii) PUBLICATION INFORMATION:
(A) AUTHORS: Kalnins et al.
(B) JOURNAL: EMBO J.
(C) VOLUME: 2 (D) PAGE: 593 (E) DATE: 1983 (iv) SEQUENCE DESCRIPTION: SEQ ID NO: 9 l 9

Claims

We Claim:

1. A composition comprising a polypeptide which contains a receptor-binding domain, a cytoplasmic translocation domain, a nuclear translocation domain, and a means for connecting a selected macromolecule to the said polypeptide.

2. The composition of Claim 1 wherein the said macromolecule is selected from the group consisting of nucleotides, oligopeptides, polypeptides, and proteins, a nucleotide sequence that encodes for factor VIII, a nucleotide sequence that encodes for alpha-1-antitrypsin, a polypeptide which is a regulator of gene expression, Beta-galactosidase.

3. The composition of Claim 1 wherein the said receptor-binding domain is a toxin-derived ligand for a specific cell receptor, is derived from diphtheria toxin, or from Pseudomonas exotoxin A.

4. The composition of Claim 1 wherein the said cytoplasmic translocation domain is derived from Pseudomonas exotoxin A.

5. The composition of Claim 1 wherein said nuclear translocation signal domain is selected from the group consisting of SV40 nucleic acid sequence, yeast alpha-2 nucleic acid sequence, and GAL-4 nucleic acid sequence .

6. The composition of Claim 1 wherein the means for connecting the macromolecule to the nuclear translocation domain is a polycationic macromolecule which is selected from the group consisting of poly-L-Lysine, poly-D-Lysine, poly NTS. ornithine, putrescine, a histone. GAL 4, a homeobox domain. spermidine. and spermine.

7. A method for inserting an exogenous macromolecule into a target cell nucleus comprising the steps of:
a) administering a polypeptide which contains a receptor-binding domain, a cytoplasmic translocation domain, a nuclear translocation domain. and a means for connecting a selected macromolecule to the said polypeptide. to target cells, b) incubating cells with said polypeptide, and c) determining transfer by an assay.

8. The method as in Claim 7 wherein the polypeptide is as described by Claim 2, Claim 3, Claim 4, Claim 5, or Claim 6.