CA2187818A1 - Gene delivery fusion proteins - Google Patents

Abstract

The invention provides gene delivery fusion protein (GDFPs) for use in gene transduction of target cells, such as mammalian cells. The GDFP contains a nucleic acid binding domain (NBD) that binds to a targeted nucleic acid to be transduced, fused to a gene delivery domain (GDD) that mediates or augments transfer of the targeted nucleic acid into the target cell. The GDD contains one or more components that facilitate gene delivery, including binding/targetting components, membrane-disrupting components, transport/localization components and replicon integration components.

Description

2Is7~la WO 95/28494 ~ '0 ~738 .

GENE DFl TVERY FUSION pRoTFr~s T~ ' I Fi~lti The invention relates to the field of gene delivery, more ~ to proteirls useful for ~UL~ ,, pul~ ' ' into target cells. Still more sy~_;rl~,dll~ the invention relates to fusion proteirls that are capable of both binding to a pol~ ' ' of interest, and of facilitating delivery of thc bound p~ uLic-il. to a target cell, especially to a ' target cell.
Back~rourld Many viruses bave been adapted for use as gene delivery vectors for cells. Viruses have bighly effcient ' for entering cells, and in some cases also have specific ' for integrating the viral genome into the host cell i ' . The high efficiency of gene t. ' afforded by the viral vectors is the principal advantage of using a virus-based system for gene delivery. In addition, the fact that the viruses are particulate allows virus-based systems to be considered for in vivo gene delivery. These attributes have led to the wide use of viral vectors in gene transfer studies. Viruses that have been used for this purpose include l~11UVU~ D, Od~lllJVUl~D~ yOl vuvul.~iC6, ~)~YUVO VUI D~D, poxviruses and L~ly~ . UhD~,D.
More recently, the utility of viral vectors bas led to the use of ICLIUVUL~D andd~.wvuuDcD in gene therapy ~
Aithough the virus-based delivery systems can give rise to high efficiency of gene delivery, they suffer from a number of ~uv ,, For example, the most widely used virai system, the retrovirai vectors, have been extensiveiy modified tû
prevent the generation of replication-competent retrovirus (RCR), but since such RCR
has the potentiai to be l ' O (see Donahue et ai., J. Exp. Med. 176:1125-1135, W095/28494 2~87~18 ~ S/0~738 .

-2-1992), all retroviral ~ICy~ iul.a for use in gene therapy must undergo extensivevalidation testing to coMIrm the absence of RCR before use. In addition to these safety concerns, retroviral and other viral vectors can place size and sequence constramts on tLle genetic material that can be transferred and on the target cells that can be infecoed S (see, e.g., Israel & Kaufman, Blood, 75:1074-1080, 1990; ~' ' & Temin, Nature 299,265-268, 1982; Soead et al., Blood, 71:742-747, 1988; and Bodine et al., Blood, 82:1975-1980, 1993).
The d.~ h)l,l...,lli of efficieM non-viral gene delivery (NVGD) systcms would allow gene L~ f../~.,..e therapy studies to be performed in the absence of the itrulr .. 1;.) ~I limitations of the viral vectors, and could also have the advantages of ease of scalability, cost and speed of generation. Based on these advantages, non-viral gene delivery systems could also allow morc- discases to he treaoed through genetherapy by making injectable gene delivery sysoems a rcality~
Existing non-viral gene delivery sysoems can be roughly divided into physical 15 and L ' approaches. The physical methods include such oechniques as d~ uy~ iu..~ particle bU..l~ ' t~ scrape loading and calcium phosphaoe (see, e.g., r. et al., P.N.A.S. 84:8463-8467, 1987; Cheng et al., P.N.A.S. 90:4455-4459, 1993; and Kriegler, M. (ed.), rGene Transfer and FYpn!ccion a Laboratory Manual,r 1990, W.H. Freeman Publishers). The 20 l~;.r 1, . ~I methods involve mixing tLle DNA to Lle delivered with reagents such as DEAE-dextran, gramicidin S, liposomes, ~ul~ ' polymers, pul~
polybrenc, cationic prooeins and poly-L-lysine-based conjugaoes (sce, e.g., Kawai &
Nishizawa, Mol. Cell. Biol. 4:1172-1174, 1984; Behr et al., P.N.A.S. 86:6g82-6986, 1989; Rose et al., P.N.A.S. r - ~ , 10:52û 525, 1991; Pardridge & Boado, F.E.B.S. Lett. 288:30-32, 1991; Legendre & Swka, P.N.A.S. 90:893-897, 1993;
Haensler & Szoka, Bioconj. Chem. 4:372-379, 1993; and Wu and Wu, J. Biol. Chem.
262:44294432, 1987).
These different approæhes vary in tneir efficiency of gene delivery and in theirability to confer long-oerm (i.e. stable) reoention of transferred sequences. However, the l.;~ h- `;. -I approaches are in general more attractive from a gene therapy point of view because such approaches have a greaoer pooential for use witLlin injectable gene delivery sySoems than do most of the physical approaches.

-WO 95/28494 2 1 ~ 7 ~ 1 8 F~l~u~,''1~4738 . ~

-3-One problem with the use of conjugates based on poly-L-lysine or other basic polymers, which are assembled via chemical cross-linking, is that the chemical steps required for cross-linking can be both imprecise and ~ . l. ..~.-.,.. Moreover, it can be very difficult to control the ~u; l.;~ y of the different conjugate . in such 5 a system, particulariy as more r ' are added to facilitate gene delivery.
Summarv ûf the InV~nrinn In Yiew of the continuing and unmet need for safe, efficient and stable non-viral gene delivery systems, the present invention provides a generalized approach for the 10 modular ~ . of fusion proteins that are capable of both binding to a ~ulyllu.l.u~ of interest, and of facilitating delivery of the bound ~ ' ' to a target cell, especially to a human target cell for gene therapy.
The proteins of the present invention, termed Gene Delivery Fusion Proteins (GDFPs) comprise a nucleic acid binding domain (NBD) tbat contains a component 15 capable of binding tbe targeted nucleic acid; fused to a gene delivery domain (GDD) tbat contains one or more, , that mediate or facilitate delivery of the targetednucleic acid to the target cell, As described in detail below, nucleic acid binding domains can comprise any of a rlumber of . . the essential feature of which is that they are capable of 20 binding nucleic acids. A rlumber of such . are known in the art (see, e.g., tbe refetences cited below), including proteins tbat bind nucleic acids in a sequence-specific marmer and proteins that bind tn~cleic acids relatively non~ ,. For purposes of discussion and '' , nucleic acid binding domains can be ., '~, grouped into either of t~vo basic subsets depending on whether the nucleic 25 acid bindmg domain does or does not contain an analog of a , ~ rl~ nucleic acid binding protein, as described in more detail below.
In a first type of gene delivery fusion protein of the present mvention (sometimes referred to herem as a "Type-l GDFP~), the nucleic acid binding domain contains an analog of a sequence-specific nucleic acid binding protein (sequence-30 specific NBP). In a second type of gene delivery fusion protein of the presentinvention (sometimes referred to herern as a "Type-ll GDFP~), the nucleic acid binding domain contains an analog of a sequence-non-specific nucleic acid binding protein

4 ~ ~ 8 7 8 1 8 ~l/u~_ 0 1738 .
(sequence-non-speciffc NBP) and does not contain aa analog of a sequenee-speeific NBP.
Thus, one ~ .o.l.~ of a GDFP of the present invention is a ~ ol.~ul~
useful in delivering a targeted nucleic acid to 2 target cell, comprising a gene delivery

5 fusion protein (GDFP), said GDFP comprising a nucleic acid binding domain (NBD) that contains a component capable of binding to a eognate r~ ~, sequenee in the targeted nucleic acid which component is derived from a sequence-speeific nucleic acid binding protein; fused to a gene delivery domain (GDD) that contains one or morethat mediate or facilitate delivery of the targeted nucleie aeid to the target 10 cell. In addition to the binding component derived from a sequenee-specific NBP, the nucleie aeid binding domain of Type-l GDFPs can also eontain additional binding -----r , as discussed below, which can h derived from either sequenee-speeifie or sequenee-non-speeifie nueleic acid binding proteins.
Another ~ ' of a GDFP of the present invention is a 15 useful in delivering a targeted nueleie aeid to a target eell, eomprising a gene delivery fusion protein (&DFP), said GDFP eomprising a nueleie aeid binding domain (NBD) that eontains a eomponent eapable of binding the targeted nueleie aeid whieh eomponent is an analog of a sequenee-non-specifie nueleie aeid binding protein; fused to a gene delivery domain (GDD) that eontains one or more , that mediate 20 or faeilitate delivery of the targeted nueleie aeid to the target eell.
In one aspeet of the invention, the , of the gene delivery domain (GDD) that faeilitate delivery of the targeted nueleie aeid to the target eell are seleeted from the group eonsisting of a ' " _~L~u~,iu~ t~ a ' ~-disrupting e~my~n~n~ a transport/~ 7~ n eomponent and a replieon integration rr~n~rnrn~
25 In another aspeet of the invention, the various funetional domains and ~ , ofthe GDFP are separated by flexible peptide linlcer sequenees (~flexons"), whieh ean er~anee the ability of the c- --~ to adopt - ~ relatively ;..-of each other.
Another ~ ' " of a GDFP of the invention is a 30 pGIrllu.l~vli.l. encoding a GDFP. In a preferred I " of this type, the pC~ JLid~ is an expression veetor and is arranged so that the various domains and - - -r of the GDFP are expressed as an in-frame fusion produet, thereby allowing WO95/28494 2 i ~ 78 1 8 1~IIU~ 38 for efficient modular synthesis of the GDFP as a single 1~ ' product. Yet another ~ ,..l,~.li -,. .ll is a method of using the above-described l~:C~
pol~ ,lile to produce a GDFP, said method comprising the steps of causing the poly"~.~.levlid- tO be transcribed and/or translated and recovering a GDFP.
5 As discussed herein, the preferred method involves the modular synthesis of the GDFP
as a single protein product. Yet another ~" l,o~ is a method of using a GDFP to deliver a targeted nucleic acid (tNA~ to a target cell, the method comprising the steps of contacting the GDFP with the targeted nucleic acid to produce a GDFP/nucleic acid complex and contacting said GDFP/nucleic acid complex with the target cell.
10 Preferably, the tNA is an expression vector. Preferably, the target cell is a "
cell. Yet another ~ 1 is a cell produced by the above-described method of using a GDFP and the progeny thereof.
Brief DescriDti~n of the Drawings Figure 1 is a schematic . of an ~ b~ ' of the Gene Delivery Fusion Protein (GDFP) concept using a Type-I GDFP.
Figures 2A and 2B are diagrams of the cloning strategy used to generate expression vectors encoding IL-2, GAL4, and the GAL4/IL-2 and IL,2/GAL4 GDFPs.
Figure 3 is an SDS-PAGE gel of 35-S labeled GAL4/IL-2m GDI P.
Figure 4 is a gel-shift assay showing retention of DNA binding activity by the GAL4/lL-2m GDFP.
Figure 5 shows retention of IL-2 bioactivity by the GAL4/IL-2 GDFP.
Figure 6 is an SDS-PAGE gel of 35-S labeled GAL4/IL-2 GDFP and IL-2/GAL4 GDFP.
Figure 7 shows sequence-specific DNA binding of the GAL4 protein and the IL-2/GAL4 and GAL4/IL-2 GDFPs.
Figure 8 shows the cytokine bioactiviy of the IL-2/GAL4 and GAL4/IL-2 GDFPs.
Figure 9 shows the results of an assay ~ the abiliy of GDFPs to bind to I1~2 receptor-bearing CTLL.

WO95128494 21 87~1 8 ~ r 1738 .

Figure 10 shows the results of an assay A, ..,....`1.,.1;,.~ the ability of GAL41~L-2 GDFP and IL-2/GAL4 &DFP to mediate binding of a target oligomer to IL-2 receptor-bearing CTLL.
Figure l l shows the results of an assay ~ t~ the ability of GAL41IL-2 5 GDFP to mediate binding of a target plasmid to IL-2 receptor-bearing CTLL.
I~P-~ DescriDtion of the Jnvrn~ n The inYention provides a non-viral gene delivery system by which DNA, RNA
and/or analogs thereof ("targeted nucleic acid" or "tNA" to be used in gene delivery) 10 are modified by association with a gene delivery fusion protein (GDFP). The non-viral gene delivery system of the present invention comprises a , -' ` complex of two separate entities: the targeted nucleic acid to be delivered, and a GDFP. The GDFP comprises a nucleic acid binding domain (NBD) that can bind to t_e targetednucleic acid and thus lead to the formation of a GDFP/tNA complex; fused to a gene 15 delivery domain (GDD) tbat cam mediate or facilitate the delivery of the GDFP/tNA
complex into the target cells.
In a preferred ~1 " of the invention the open reading frames encoding the Yarious GDFP domains and -- q~ are fused to enable expression of ihe GDFP as a single ~ul~ii~. However, the GDFP may aiso comprise, for example, 20 one or mote short flexible peptide linker sequence (~flexons") between the mdividual domains and/or ---r t'~n''~l r The terms ~I,u..~ , "peptide" and "protein~ are used ' ~ o 25 refer to Dolymers of ar~ino acids and do not refer to any particular lengths of tile polymers. These terms also include post- ' '5~ modified proteins, for example, 81~v~' d, acetylated, I ' ,' .~' ' proteirls and the like. Also included within the definition are, for example, proteins containing one or more arialogs of an amino acid (including, for example, unnatuIal ariuino acids, etc.), proteins with 30 substituted linkages, as well as other i---~l;l; -1--....~ known in the art, both natilrally occurrmg and non-naturally occurring.

2~878~8 W0 95/28494 r~ 738 "Native" pol~ or polyl.u~ ,Li~ refer to polypeptides or polyl.~ ,Lid~ recovered from a source occurring in nature. Thus, the phrase "nalive viral binding proteins" would refer to naturally occurrmg viral binding proteins.
"Mutein" forms of a protein or pol.~ id. are those which have minor 5 alterations in amino acid sequence caused, for example, by site-specific ~ or other ~ by errors in ~ r '' or translation; or which are prepared a~llLII.,.i.~.ll.y by rational design. Minor alterations are those which result in amino acid sequences wherein the biological activiq of the pul.~ JLid~. is retained and/or wherein the mutein pol~ ide has at least 90% homology with the natiw form.
Arl "analog" of a yuly~ ; X includes fragments and muteins of p~
X that retain a particular biological activiq; as well as p~l.r~.,Jli.l-, X that has been iU~UI~ ' ' into a larger molecule (other than a molecule within which it is normally found); as well as synthetic analogs tnat have been prepared by rational design. For example, an analog of a DNA binding protein might refer to a portion of a native DNA
15 bindmg protein that retains tbe abiliq to bind to DNA, to a mutein thereof, to an entire native binding protein that has been --- . ' into a fusion protein, or to an analog of a native binding protein tnat has been a.~h..~_ti~ prepared by rational design.
Ilr~ n refers to a polytneric form of nucleotides of any length, either , ;1 .. .- - 1. .,1 ;.1. or .1~ ' ' or analogs thereof. This term refers only to the primary sttucture of the molecule. Thus, double- and smgle-stranded DNA, as well as double- and single- stranded RNA are included. It also includes modified ~vlJ ' such as methylated or capped PUIJ
An "analog~ of DNA, RNA or a p.,l.~ ' ' . refers to a resembling naturally-occurring p~ ' ' in form and/or function (~ , in the abiliq to engage in sequence-specific hydrogen bonding to base pairs on a ~ .... 1-1- " - - .r pul.yl.~l~,~/Lid~ sequence) but which differs from DNA or RNA in, for example, the possession of an unusual or non-natural base or an altered backbone. A
large varieq of such molecules have been described for use in antisense: ' ' ,,y, see, e.g., E. Uhlmann et al. (1990) Chemical Reviews 90:543-584, and the .,.. reviewed therein.

wo 9s/28494 2 1 8 ~ 1 8 . ~u~ ~173~
An "antisense" copy of a particular ~O~ VP~LIC refers to ~ y sequence that is capable of hydrogen bonding to tbe paly..u,,l~vli.lc and may, therefore, be capable of modulating expression of the ~ulyl.~ vlide (i.e. by "antisense"
regulation). Such an antisense copy rnay be DNA. RNA or analogs thereof, including S analogs haYing altered backbones, as describe~ above. The ~ul~ v~idc to which the antisense copy binds may be in single-stranded form (such as an mRNA molecule) or in double-stranded form (such as a portion of a I-.u.,-v~v~).
A "replicon~ refers to a ~ulr ' comprising an origin of replication (generally referred to as an sri sequence) which allows for replication of the 10 p~ vlidc in an appropriate host cell. Examples mclude replicons of a tatget cell into which a desired rlucleic acid might integrate ~in particular, nuclear and ,Lulllu~u~j, and also e.~u.~,L~ ' replicons such as plasmids).
"r~ t," as applied to a p~l~ ' ', means tnat the P~IJ ~ ~ is the product of Yarious ' of cloning, restriction and/or ligation steps 15 tesulting in a construct that is distinct from a p~ vlid~ found in nature.
~R~"....I. ~ may also be used to refer to the protein product of a P~l.r ' ' Typically, DNA sequences encoding the structural coding sequence for, e.g., ~ , of the NBD and GDD, can be assembled from cDNA fragmerlts and short li" ' ' linkers. or from a series of 'i" ' ' . to prsvide a 20 synthetic gene which is capable of bemg expressed when operably linked to a ' regulatory region. Such sequences are preferably proYided in the form of an open readirlg frame I .,~1 by mternal non-translated sequences (i.e.
"introns~), such as those commonly found in eukaryotic genes. Such sequences, and all of the sequences referred to in the context of thc present invention, can also be 25 generally obtained by PCR I I;r, -:; ,-- using viral, ~IVL~ U~i~ or eukaryotic DNA or RNA templates in: ~ with appropriate PCR a~nplimers.
A .c~ expression vector" refers to a povl~.lu.l~vLidc which contains a ~, ,,. .~, . ;1~1~. -I regulatory region and coding sequences necessary for the expression of an RNA molecule and/or protein and which is capable of being introduccd into a target 3û cell (by, e.g., vir I infection" r '' , e~ llu~uul~l iun or by the nsn-viral gene delivery (NVGD) techniques of the present invention). A fur~her example would be an expression vector used to express a GDFP of the present invention.
. , . . .. . . . _ _ _ _ . = _ _ _ _ _ W09S128494 21 8 ? 8 ~ 8 r~l"J~,~ 1738 _9 _ "Rc-c,.. l. - .l host cells", "host cells", "cells", "target cellsn, "cell lines", "cell cultures", and other such terms denote higher eukaryotic cells, most preferably ".- ,. .:~1; cells, which can be, or haYe been, used as recipients for Ir~
vectors or other transfer poly.. ! .. 1;.1. `, and include the progeny of the original cell S which has been transduced. It is understood that the progeny of a single cell may not necessarily be compleely identical (in ~lul~ lo~;y or in genomic or total DNA
.:~.. 1.l.. :) to the original parent cell, due to natural, accidental, or deliberate mutation.
An "open reading frame" (or "ORF") is a region of a pG;J ' ' sequence 10 that can encode a PUI~ ;~ or a portion of a pol~ id~ (i.e., the region may represent a portion of a protein coding sequence or an entire protem coding sequence).
"Fused" or "fusion" refers to the joining together of two or more elements, . etc-, by whatever means (mcluding, for example, a "fusion protein" made by chemical , (whether covalent or non-covalent), as well as the use of am 15 im-frame fusion to generate a "fusion prooemr by ' means, as discussed infra). An "in-frame fusion" refers to the joining of two or more open reading frames (ORFs), by ' means, to forln a single larger ORF, in a manner that maimtains the correct reading frame of the original ORFs. Thus, the resulting fusion protem is a smgle protein containing two or more segments that 20 correspond to ~ encoded by the original ORFs (which segments are not normally so joined in nature). Although the reading frame is thus made continuous throughout the fused segments, the segments may be physically separated by, for example, in-frame flexible ~ Jli.le linker sequences ("flexons~), as described infra.
A ~flexon~ refers to a flexible pul~lJtid. Iinker sequence (or to a nucleic acid25 sequence encoding such a pol~ id.) which typicaTly comprises amino acids having small side chains (e.g., glycine, alanine, valine, leucine, isoleucme and serme). In the present invention, flexons can be ~ . ' in the GDFP between one or more of the various domairls arld ~ T . - _ flexons bet veen these , is believed to promote rul~liu~l;i~ by allowing them to adopt ~ relatively 30 ;...l. l,....l. ~l~ from each other. Most of the ammo acids r ' ~ into the flexon ~vill preferably be amino acids having small side chains. The flexon will preferably comprise bet~veen about four and one hundred amino acids, more preferably between . 2~87~
WO 95128494 r~ o.. , _.'C 1738 .

about eigh~ and fifty amino aeids, and most preferably bet veen about ten and thirty amino acids. Flexon ("Pixy") sequences described in U.S. Patents S,0737627 and 5,108,910 will also be suitable for use as flexons.
A !~ rjc~ ~ regulatory region" or "1.. ~ ' eontrol region" refers to 5 a ~rCrl.~ uLid~ , all of the cis-acting sequences necessary for ..~. .;1.;;...,, and may include sequenees necessary for regulation. Thus, a ' regulatory region includes at least a promoter sequenee, and may also inelude other regulatory sequences such as enhaneers, I.. -- ;1~ -- factor binding sites, pcil.~aJc~ l~iiu~L signals and splicing signals.
"Operably linked" refers to a j~ wherein the ~ , so described are in a 1~' ' ,. permitting them to funetion in their intended manner. Fûr instance, a promoter sequenee is operably linked to a coding sequenee if the promûter sequence promotes i of the coding sequenee.
"T ' 1," as used herein, refers to the ill~ ' of an exogenous 15 ~1~ ' ' into a host eell, i..~ , of the method used for the insertion, whieh methods inelude, for e~ample,; r ' , viral infeetion, i r...d~;.... and the non-viral gene delivery teehniques of the present invention.The introduced polJI.~~ Li.le may be subly or transiently maintained in the host eell.
Stable typieally requires tbat the introdueed pul~ ' ' eitber eontains 20 an origin of replieation compatible with the host cell or integrates into a replicon of the host eell such as an "rllldDu~l replicon (e.g. a plasmid) or a nuelear or .. . .. .
~ l~e~luvil,~,'' are a elass of viruses whieh use R~A-direeted DNA ~ul~...~,~, or reverse ~ - . to replicate a Yiral RNA genome resulting in a double-stranded 2~' DNA ' whieh is hl~ul~ ' into ~ ', ' DNA of an avian or ". 1: -- host cell. Many sueh ~UUVil~_i. are Icnown to those skilled in the a~t and are deseribed, for example, in Welss et al., eds, RNA Tumor Vin~c~-c 2d ed., Cold Spring Harbor, New York (19a4 and 1985). Plasmids containing retroviral genomes are also widely available, from the American Type Culture Collection (ATCC) and 30 other sources. Tbe nucleic acid sequences of a large rlumber of these viruses are known and are generally available, for example, from databases such as GENBANEC.

218781~
W0 95/28494 r~ 738 A "sequence-specific nucleic acid binding protein" is a protein that binds to nucleic acids in a sequence-specific manner, i.e., a protein that binds to certain nucleic acid sequences (i.e. "cognate recognition sequences", infra) with greater affiniy than to other nucleic acid sequences. A "sequence-non-specific nucleic acid binding protein" is 5 a protein that binds to nucleic acids in a sequence-non-specific manner, i.e. a protein that binds generally to nucleic acids.
A "cognate" receptor of a given ligand refers to the receptor normally capable of binding such a ligand. A "cognate" recoglution sequence is defined as a nucleotide sequence to which a nucleic acid binding domain of a sequence-specific nucleic acid 10 binding protein binds with greater affiniy than to other nucleic acid sequences. A
"cognater interaction refers to an ;,.~ association based on such ypes of binding (e.g. an association between a receptor and itD cognate ligand, and an association between a sequence-specific nucleic acid binding protein and itD cognate nucleic acid sequence).
"Gene delivery" is defned as the i ~ ;.- of targeted nucleic acid into a target oell for gene transfer and may encompass i ,, " " ~, uptake, transport/lnr~':7~inn, replicon integration and e~pression.
"Ly , ' ~D" as used herein, are spherical cells with a large round nucleus (which may be indented) and scany cytoplasm. They are cells that s, recogni_e and respond to non-self antigens, and are responsible for d,,~lu~ ~ ofspecific immuniy. Included within ~ ' are B-l~ ~D and T-ly . ) ~D of various classes.
~Ly . ' ' , stem cells" are cells which are typically obtained from the bone rnarrow or peripheral blood and which are capable of giving rise, tbrough cell division, to any mature cells of the Iymphoid or I ~ systems. This term includes committed progenitor cells with significant though limited capaciy for self-renewal, as well as the more primitive cells such as those capable of forming spleen colonies in a CFU-S assay, and still more primitive cells possessing long-term and/or ' ' ~ re-populating abiliy in a i .' " host.
~Ly . ' . cells~ include the various mature cells of the Iymphoid or I . systems (including Iy .' ~D and other blood cells), as well as Iy . ' ' , stem cells.

2 1 878 1 ~
w0 95128494 P~ 738 A "primary culture of cells" or "primary cells" refer to cells which have been derived d~rectly from in yivo tissuç and not cxtensively passaged. Primary cultures catl be .1;~1;.,~...~1.. d from cell lines and established cultures principally by the retention of a karyotype which is substansial~y identical to the karyotype found in the tissue from which the culture was derived, and by the cellular responses to ,~ of the e.lvi.v..~ ,..t which are ~ub~ similar to the ill viw cellular responses.
As is described in detail below, the non-Yiral gene delivery complexes of the present invention comprise gene delivery fusion proteins (GDFPs) that bind targe~ed nucleic acid through a nucleic acid binding domain tNBD) and facilitate gene delivery 10 through a gene delivery domain ~GDD). Each of these domains can comprise a number of different functional ~ t~ and sub~ Some of these potential r".. l.. ~ t` are ' in the following list:
NON-~IRAI, GEN~ Dhl,lVERY COMPT ~X (~he "GDPPltNA Comvle%"l 1, Gene ~ rer~ ion Protein ~GDFP) A. Nucleic Acid Binding Domain (NBD) (1) Nucleic Acid Binding tNB) component (2) Otherpossible ~ (e.g. mediating c~ of tNA) 20 B. Gene Delivety Domain ~GDD) (1) BindinglTargeting (B/'l-) component -Disrupting (M-D) component (3) TransporVT ~l~li7:~tjf~n (T/L) component (4) Replico~ Integration (Rl) component 2, Ts~ Eeted Nucleic Acid (tNA) A. Binding sites for the GDFP (see infra) B. Sequence of inurest (e.g. gene to be delivered) C. Other possible sequences (e.g. selectable markers) Each of these domains and ~ . as well as additional elements that may be included, are deflned and described in detail below.

W095128494 2 1 8 7~ 1 ~ p~usg5~o4738 The practice of the present invention will employ, uniess otherwise indicated, anumber of .V~ iUI~I techniques of molecular biology, ~ .ul ;ùlu~;y, DNA, and i..ll...~..olo~y, which are within the sicill of the art. Such techniques are explained fully in the literature, see, e.g., Kriegler, M. (ed.), "Gene Transfer and 5 FYrrl~ccion, a Laboratory Manual," (1990), W.H. Freeman Publishers; Sambrook, Fritsch, and Maniatis, "Moiecular Cloning: A Laboratory Manuai," Second Edition (1989); F.M. Ausubel et al. (eds.), "Current Protocols in Molecular Biology," (1987 and 1993); M.J. Gait (ed.), "O' ,, ' ' Synthesis," (1984); R.l. Fresimey (ed.), "Animal Cell Culture," (1987); J.M. Miller and M.P. Caios (eds.), rGene Transfer10 Vectors for ~rr~ n Ceiis," (1987); D.M. Weir and C.C. Blacicweii (eds.), "Handbook of F.I.. ;.. ~ l T ' _~;" J.E. Coligan, A.M. ~ruisbeek, D.H.
Margulies, E.M. Shevach and W. Strober, (eds.), "Current Protocols in r ~ _y,~
(1991); and the series entitied ~Methods in El~ l.lùlG~,r (Academic Press, inc.). Aii pater~ts, patent ~ , and r ' ' - mentioned herein, both supra and irlfra, are hereby i.~ ' herein by reference.
~a~ons of TvDe I Gene Deli~ery Fu~sion Pro~e~
Til~ Gene Deiiverv F -cinn Protein / Tar~eted nucleic acid Cn~ py (GDFP/tNA) One concept of the preserlt invention is to create ' gene delivery fusion proteins (GDFPs) that are able to bind to a cognate l~ _ sequence in a targeted r~ucleic acid (tNA) and faciiitate delivery of the tNA into a target oell. The GDFPs bind targeted nucleic acid through a nucleic acid binding domain (NBD) andfaciiitate gene delivery through a gene delivery domain (GDD).
Thus, in the context of the present invention, targeted nucleic acids can be delivered via one or more steps that are mediated or augmerlted by GDFPs. In particuiar, the gene delivery process can include one or more of the following steps:
(1) binding and/or targeting of the GDFP/tNA complex to tile surface of a target oell;
(2) uptake of the tNA (with or without the GDFP) by the target cell; (3) in~rrll ' transport and/or IGcalization of the tNA to an organelle such as a nucleus or ' ' and (4) integration of the tNA into a ceiiuiar replicon such a i~ul,luav...e. A particular GDFP need not necessarily perform ail of these functions.

W0 95/28494 2 t ~ 7 ~ l ~ r~ s ~0 ~738 For example, a GDFP inter~ded to deliver an expression vector to the nucleus of a cell could be constructed to contain: ~i) an NBD capabie of binding to a cognate recogiution sequence on the expression vector and; ~ii) a GDD having oniy a transportl1rr~li7~rinn component such as a nuclear Inr~li7:~inn sequence. S~ich a GDFP couid then be 5 complexed wit'ii targeted nucleic acid and introduced iiito target ceiis by a ~
method such as ~l~lluyu~aliull. The GDFP would then facilitate transportl~ 7~rinn to the nucleus, perh~ips to a specifc site in a replicon, aiid tnus erlhance expression of the vector. Al~eriiatively, foi example, tiie ~ru~c~,Ai~ i GDD couid be modified to include a ' ' ' ~'~.ill~ component and a ...~ disrupting ~ L ' Usilig 10 such a GDD, tne GDFP/tNA complex couid be directed to a particu'iar cell tvpe within a population of cells, and uptai~e of t~ie complex could proceed without t~ie nced for, e.g., ~ L~ Use of thc GDFPs in ~ ' witli techniques such as UyUl.lliU,I, as in t~ie former example, would of co~irse be more a~,L,., . for in vitro genc de'iiveiy. Use of GDFPs as described in t~ie latter exarliple could i~e readily 15 applied to the delivery of gelies eitbier in vitro or in vivo. Sir~iiiarly, the GDFP/tNA
complexes couid be used as admixtures with other proteins or simple chemicais that enhance gene delivery. This could include, for examp~e, enhancu~g the uptake of GDFP/tNA comple~es by adding membr~ine ~iisrupting agents ~; tran~.
Other ~ of ~ - can be prepared (and pârticuiar versions of 20 the ~ can be selected) according to the specific design objecdves of the genedelivery scheme. These objectives include, for example, the locadon of tile celis to i~e targeted, the desired celluiar specificity of targeting, and the desired sub-celluiar destination of tile tNA.
The individual domairls and ~ of the GDFP/tNA complex and their 25 ~ and assembly are described in more detai'i below.
1. The Gene Deliverv Fusion Protein (GDFP~
The GDFP comprises t vo inajor domailis, a nucleic acid binding domain (NBD) and a gene delivery domain ~GDD). Each of these major domailis comprises orle or30 more ~ t~ fæilitating nuc~eic acid binding and gene delivery, r~
'rhesè individual ~ , may be derived from naturally-occurring proteins, or they riay be synthetic (e.g. an ana'iog of a natura'ily-occurring ~ Typicaliy, _ WO9~128494 2 1 8 7 8 1 ~ r~ o4738 cloned DNA encoding various ~ will already be available as plasmids -aldhough it is also possible to syndhesize pol~ ' ' encoding the r~
based ~lpon published sequence il~llll.~Liu~. rul.~ lcv~ encoding dhe cu...~
can also be readiiy obtained using pol~....l."~ chain reaction (PCR) ' '-'~Oy, as S described, for example, by Mullis and Faloona (1987) Meth. ~.~.yIIIUI~l;J 155:335.
In the Cu.~.lu liu.. of dhe GDFP, discussed in mote detaii below, DNA
sequences encoding dhe domains and dheir various . . are preferably fused in-frame so that dle GDFP can be u..~ Li.~ syndhesized as a smgle pUl.~Lid cham (i.e. not requiring further assembly). The various domains and ~ , can 10 also be separated by flexibie peptide linicer sequences cailed "flexons~ which are defned m more detaii above.
A. The Nucleic Acid p:nrlin~ Domain (NBD) A nucleic acid bmding domain is a length of p~ ~"li~ capable of binding 15 (eidher directdy or indirecdy) to dhe targeted nucleic acid (dNA) widh an affnity adequate to allow the gene delivery domain of the GDFP to mediate or augment dhedelivety of the tNA mto a targe~ cell. Most ~U.A.~ " dhe NBD will bind direcdy to tile tNA without the need for any " y binding element.
In Type-I GDFPs, the NBD contains a sequence-specific binding component dlat 20 is an anaiog of a sequence-specific nucleic acid bindmg protem. In one preferred ,. ~.~1 -- ..~ of this type, the component ailows the nucleic acid binding by the NBD to be sequence-specific with respect to the dNA, in which case the NBD may bind to a specific cognate recogmtion sequence within the tNA; as is iilustrated in Figure 1.
As described herein, one particular advantage of dhe Type I GDFP approach is that it not oniy allows the ' attachment of delivery , to the tNA, but also aliows the GDFP to be positioned at pre-determined locations with respect to the tNA. For example, dhe positioning of NBD cognate ~ v -- sequences m proximity to terminai mtegrase recognition sequences can faciiitate thc use of GDFPs to mediate mtegration, as described below.
The NBD may comprise, for example, a icnown nucleic acid binding protein, or a nucleic acid binding region thereof. The NBD may also comprise two or more nucleic acid binding regions derived from dhe same or different nucleic acid binding woss/z8494 2 1 8 7~ t 8 l~ Ot738 -lo-proteins. Such ,. '~ . of nucleic acid binding regions in the NBD can allow for the interaction of the GDFP with the targeted nucleic acid to be of desirable specificity and/or higher affinity. This strategy can be used alone or in . ~
with ~ of recognition sequence motifs in the tNA to increase bindmg 5 avidity, as discussed below.
DNA encoding the NBD domain of the GDFP may be obtained from many different sources~ For example, many prouins that are capable of binding nucleic acid have been . ' ' '~ cloned and their cognate target l~ ,, ` ` sequences have beenidentified (see, e~g~, Mitchell & ijian, Science 245:371-378, 1989; Pabo & Sauer, Ann. Rev. Biochem~ 61:1053-1095, 1992; Harrison, S C., Nature 353:~15-719, 1991;Johnson & McKnight, Ann. Rev. Biochem. 58:799-839, 1989; and references reviewedtherein, hereby illI,UI, ' ' by reference?. Such sequence-specific binding proteins include, for example, regulatory proteins such as those involved in ~ ûr nucleic acid replication, and typically have a modular: ` consisting of distmct DNA binding domains and regulatory dûmains ~see, e~g., Struhl, Cell 49:295-29~, 1987; Frankel arld Kim, Cell 65:717-719, 1991; and Pabo & Sauer, Ann. Rev~
Biochem. 61:1053-1095, 1992; and references reviewed tberein, hereby `
by reference}. A number of families of such nucleic acid binding proteins have been . 1 ," .. . ;,. .1 on the basis of recurring structural motifs including, for example, 20 Helix-Turn-Helix proteins such as the I.~ ~ ; y,~ c~ Iatnbda cI repressor;
T~ ' proteins such as the DrosoDhila A ~ ' regulator; the POU
domain present m proteins such as the ' i , factor Oct2; Zinc fung proteins (e.g. GAL4); steroid receptors; leucine zipper proteins (e.g. GCN4, C/EBP and c-jun); beta-sheet motifs (e.g. the ~ al.yuLic Arc reptessor); and other 25 families (including serum response factor, oncogenes such as c-myb, NFkB and rel, and others); see, e.g., Pabo & Sauer, Ann. Rev. ~iochem. 61:1053-1095, 1992, andreferences reviewed therein, hereby ~ ,UI~ ' ' by reference.
For many of these proteins, the nucleic acid binding domains have been mapped in deuil; and, for a number of such domains, ' fusions with Lt~ ~ul~u....
30 sequences have been made and shown to retain the bmdmg activities of the parenul DNA binding domain. For example, in the case of the yeast-derived ~
activator GAL4, the DNA bindmg domain has been deftned, and fusions of this domain _ . _ _ , _,,, ,, ., ,,, .. , , ., . ,, . . . , , .. ,,, .,,, ,, ,,, ,,,,, ,,, ,,, ,,,, _,, _ WO95/28494 2: 8 7~ ~ 8 ~ 738 to II.,t~,.ulu~uu~ adjoining sequences have been made that retain DNA sequence-specific binding activity (Keegan et al., Science 231:669-704, 1986; Ma & Ptashne, Cell 48:847-853, 1987). This ability to functionally "swap" binding domains has also been shown for a number of other DNA binding proteins, including, for example, the E. coli S lex A repressor (Brent and Ptashne, Cell 43:729-736, 1985), the yeast ~
activator GCN4 (Hope and Struhl, Cell 46:885-894, 1986), the ' ,' _ lambda cI repressor (Hu et al., Science 250:1400-1403, 1990), the factors Spl (Kadonaga et al., Cell 51:1079-1090, 1987) and C/EBP (Agre et al., Science 246:922-926, 1989). Similarly, functiûnal swapping has been reported m the nuclear DNA-binding steroid hormone receptûrs (see, e.g., Green and Chambon, Nature 325:75-78, 1987). See also, e.g., EUug & Rhodes, Trends Biochem. Sci.
12:464-471, 1987; Berg, Cell 57:1065-1068, 1989; Wasylyk et al., Eur. J. Biochem.
211:7-18, 1993; Faisst & Meyer, Nucl. Acids Res. 20:3-26, 1992; Struhl, Trends Biochem. Sci. 14:137-140, 1989; and Nelson & Sauer, Cell 42:549-558, 1985.
Sequence-specific rlucleic acid binding proteins can exhibit a range of binding affinities to different cognate nucleic acid sequences in vitro (see, e.g., Vashee et al., J.
Biol.Chem 268:24699-24706, 1993).
Virally encoded nucleic acid bindmg proteins can also be used in the present invention. These include, for example, the adenovirus E2A gene product, which can bind single-stranded DNA, double-stranded DNA and also RNA (Cleghon et al., Virology 197:564-575, 1993, and references cited therein); the retroviral IN proteins (Krogstad & Cbampoux, J. Virol 64: 2796, 1990); the AAV rep 68 and 78 proteins (Owens et al., J. Virol 67: 997, 1993); and the SV40 T antigen (Arthur et al., J.
Virol., 62:1999-2006, 1988). The cellular p53 gene produc~, which binds T antigen, is also a DNA binding protem (Funk et al., Mol. Cell Biol., 12:2866-2871, 1992).
Similarly, RNA binding proteins have been identified and their inclusion in the NBD would associate the GDFP with a targeted RNA and thereby achieve RNA
delivery mediated by the gene deliverv domain of the GDFP. RNA binding prûteins that cam be used in the context of the present invention imclude, for example, the Tat and Rev proteins of HIV; see, e.g., Tiley et al., P.N.A.S. 89:758-762, 1992; amdCullen et al., Cell 73:417-420, 1993. Similarly, celiular RNA binding proteins, such WO 9~i/28494 r~ 5'01738 .

as the interferon-inducible 9-27 gene product ~('., - - ,--' -' i~ et al., Science 259:1314-1318, 1993), can also be used.
Nucleic acid binding domains of Type-I GDFPs can also contain (in addition to a ComponeM derived from a sequence-specific nucleic acid binding protein) one orS more CU__T that are derived from sequence-non-specific nucleic acid binding proteins. Such sequence-non-specific binding prooeins include, for example, histones (von Holt, Bioassays 3:120-124, 1986; Rhodes, Nucleic Acids Res. 6:1805-1816, 1979; Rodriguez et al., Biophys. Chem. 39:145-152, 1991); proteins such as nucleolin ~Erard et al., Eur J. Biochem. 191:19-26, 1990); polybasic ~ J~Li~ sequences such as poly-L-lysine (Li et al., R~ , 12:1763-1772 1973; Weiskopf and Li, Biul~ulJ 16:669-684, 1977), avidin (Pardridge & Boado, F.E.B.S. Lett. 288:30-32,1991); the non-histone high mobility group proteins and other proteins (see, e.g., Pabo & Sauer, Ann. Rev. Biochem. 61:1053-1095, 1992, and references reviewed therein);
that interact non-specifically with nucleic acids. Other proteins binding nucleic acid in IS a sequence-non-specific fashion mclude retroviral ~ (NC~ proteins (see, e.g., Gelfand et al., J. Biol. Chem., 268:184S0-18456, 1993).
B. The ~TPn~ Deliverv ~ (GDD) The GDD portion of the GDFP contains one or more p~ regions that 20 mediate or augment the efficiency of gene delivery. Such sequences may include, for example, ' ' _ ', _ ~ -disrupting;
ulJ~ -- -- - r ', and replicon integration , . as discussed below.
A particular GDD need not contain a component .~ L, each of the 2S ~r "~ types. Conversely, a GDD may CoMain more than a single component of a given type to obtain the desired activity. Moreover, a particular segment of a GDD might serve the function of two or more of these . , For example, a single region of a pul~,ti ic might function both in binding to a cell surface and in disruption of the membrane at that surface.

WO9517,S494 2 ~ 8 7~ I ~ r~l~v~.~o,738 (I) Rimlin,p/Tar~etiny (BIT) r~mr- n~ s Bi.~i..g/~l~ are regions of p~ hl~ that mediate binding - to cellular surfaces (which binding may be specific or non-specific, direct or indirect).
Any protein that can bind to the surface of the desired target cell can be employed as a 5 source of BIT ~ Such proteins include, for example, ligands such as cytolcines that bind to particular cell surface receptors, arltibodies, lectins, viral binding proteins, cellular adhesion molecules, and any other proteins that associate with cellular surfaces. The "receptorsr for these bmding proteins include but are not limited to proteins. MoreoYer, the receptors may, but need not, be specific amd/or restricted to 10 certain cell types. Essentially, the B/T c~ can be prepared from any ligand that binds to a cell surface molecule.
By way of illustration, one group of proteins from which the B/T
can be derived are cytokines. Cytokines are iu~., ' signalling molecules, the best ~cnown of which are involved in the regulation of " somatic cells. Several 15 families of cytokines, both growth promoting and growth inhibitory in their effects, have been ~1, . h . ;.. 1 Thus, a B/T component can comprise an amino acid sequence containing at least that portion of a cytokine p~ iL that is required for binding to receptors for the cytokme on the surface of ' cells, or a mutein of such a portion of a cytokine ~ u l~. A B/T component derived from a cytokine 20 can, but need not, also contain the portion of the cytokine tbat is involved in "cytokine effector activity, n as described below.
Examples of cytokines tbat can be used in the present mvention include, for exatnple, ' (such as 1~1~, IL,1,~, IL-2, IL-3, IL4, 1~5, IL 6, IL 7, IL-9 (P40), IL,10, IL-11, 1~12 and IL-13); CSF-type cytokines such as GM-CSF, G-CSF, 25 M-CSF, LIF, EPO, TNF-~ and TNF-,~); interferons (such as IFN-cY, IFN-,~, IFN~r);
cytokmes of the TGF-,~ family (such as TGF-,~1, TGF-,~2, TGF-,B3, inhibin A, inhibin B, activin A, activin B); ~ factors (such as NAP-1, MCP-1, MIP-1~, MIP-l,B, MIP-2, SIS,B, SIS~, SIS~, PF 1, PBP, rIP-10, MGSA); growth factors (such as EGF, TGF-IY, aFGF, bFGF, KGF, PDGF-A, PDGF-B, PD-ECGP, INS, IGF-I, 30 IGF-II, NGF-,B); c~-type intercrme cytokines (such as 1~8, GRO/MGSA, PF-4, PBP~CTAP/,BTG, IP-10, MIP-2, KC, 9E3); and ,~-type intercrme cytolcines (such asMCAF, ACT-21PAT 7441G26, LD-781PAT 464, RANTES, G26, 1309, JE, TCA3, , . . , . ... , .. . . _ _ _ W095128494 2 1 ~ 78 ~ /u.,,''0~738 MlP-ltY"~, CRG-2~. A number of other cytokines are also known to those of ski~l' in the art. The sources" ~ . targets and effector activities of these cytokines have been described and, for many of the cytokines, the DNA sequences encodmg the molecules are also known; see, e.g., Van Snick, J. et al. (1989) J. Exp. Med. 169:
363-368; Paul, S.R. et al. (1990) Proc. Natl. Acad. Sci. USA 87: 7512-7516; Gately, M.K. et al. (1991) J. Immunol. 147: 874-882; Minty, A., et al. (1993) Nature 362:
248; and the reviews by Arai, K., et al. (1990) Annu. Rev. Biochem. 59:783-836; and Oppenheim, J.J., et al. (1991) ADnu. Rev. Immunol. 9:61748; Waldman, T.A. (1989)Annu. Rev. Biochem. 58:8~5-911; Beutler, B., et al. (1988) Annu. Rev. Biochem.
57:505-18; Taniguchi, T. (1988) Annu. Rev. Immunol. 6:439-64; Paul, W.E. et al.,(1987) Annu. Rev. Immunol. 5:429-59; Pestka, S. et al., (1987) Annu. Rev. Biochem.
56:727-77; Nicola, N.A. et al. (1989) Annu. Rev. Biochem. 58:45-77; arld Schrader, J.W. (1986) Annu. Rev. Imm~mol. 4:205-30; and the particular p..hl~ reYiewed and/or cited therein, which are hereby in~l ~ ' by reference in their entirety.
15 Mamy of the DNA sequences encoding cytokines are also generally available from sequence databases such as GENBANK. Typically, cloned DNA encoding such cytokines will aheady be available as plasmids - although it is also possiole tosynthesize polJ~ .ti~D encoding the cytokines based upon the published se~uenoe r~ encoding the cytokines can also be obtDined using 20 polymerase chain reaction (PCR) . :h ~ as described, for example, by Mullis and Faloona (1987) M~th. E~ 155:335. The detection, ~ and -- of cytokines, including assays for identifyinO new cytokines effective upon a given tarOet oell type, have also been described in a number of L
including, e.g., Clemens, M.J. et al. (eds.) (1987) ~L~yL~i~J and Interferorls,~25 IRL Press, Oxford; and l~eMaeyer, E., et al. (1988) "Interferons and Other Regulatory Cytokines, " John Wiley ~ Sons, New York; as well as the referenoes referred to above.
The ligands suitable for targetmg a particular sub-population of cells will be those which bind to receptors present on cells of that sub-population. Again, taking 30 cytokines as an example, the target cells for a large number of these molecules are aheady known, as noted above; and, in many cases, the particular cell surface receptors for the cytokine have already been identified and ~ A- ~ , see, e.g., the _ _ . .. . ... . _ . . . _ w0 95/28494 2 1 8 7 8 7 ~ P~l/u~ 738 `

referred to above. Typically. the cell surface receptors for cytokines are L~ - æly.u~ that consist of either a single chain pûlypeptide or multiple protein subunits. The receptors generally bind to their cognate ligands with high affinity and specificity, and may be widely distributed on a variety of somatic 5 cells, or quite specific to given cell subsets. The presence of cytokine receptors on a given cell type can also be predicted from the ability of a cytokine to modulate the growth or other ~ of the given cell; and can be ~ for example, by monitoring the binding of a labeled cytokine to such cells; and other techniques, as described in the references cited abûve.
Thus, for example, a large number of cytokine receptors have been J and many of these are known to belong to receptor families which share similar structural motifs; see, e.g., the review by Miyajima, A., et al., Ann. Rev.
Immunol. 10:295-331 (1992), and the l ' reviewed therein, hereby by reference- Type-l cytokine receptors (or I ~ growtb factor receptors) include, for example, the receptors for IL-2, IL-3, IL4, IL-5, IL,6, ILr7, GM-CSF, G-CSF, EPO, CNTF and LIF. Type-lI cytokine receptors include, for example, the receptors for IFN-~Y, IFN-,B and IFN~y. Type-m cytokine receptors include, for example, the receptors for TNF-~, TNF-,~, FAS, CD40 and NGF. Type-IV cytokine receptors ( ~ Iike, or rlg-like," receptors) include the receptors for IL,1; and tbe receptors for IL-6 and G-CSF (which have Ig-like motifs in addition to tbe Type-l motif). Tbese receptor families are described for example, in Smith et al., Science 248: 1019-1023, 1990); Larsen et al., J. Exp. Med., 172: 1559-1570, 1990); McMahan et al., EMBO J. 10:2821-2832, 1991); and in tbe reviews by Cosman et al., Trends Biochem Sci 15: 265-269, 1990); and Miyajima, A., et al., Ann. Rev. Immunol. 10:295-331 (1992), and the L ' reviewed tberein, all of wbich are bereby i.l~ul~ ' by reference. As new cytokines are ~ ; l tbese can be employed in tbe present invention as long as tbey exhibit the desired binding ~1. ,.. r. .;~ and specificity. Tbe ;,l ..~ ;.- and .~ of cytokines, -4nd tbe use of assays to test tbe ability of cytokines to activate particular target cells, are 30 known in the art; see, e.g., Clemens, M.J. et al. (eds.) (1987) "L~, ' ' and l~.f~l, " IRL Press, Oxford; and DeMaeyer, E., et al. (1988) rlnterferons and wo ssn8494 2 ~ g 7 ~ 1 8 l ~ ~ 0 1738 Other Regulatory Cytokines." John Wiley & Sons, New York; as well as the references referred to above.
The choice of a particular ligand will depend on the presence of cognate receptors on the desired target cells. It may also depend on the ~ e absence of cognate receptors on other cells which it rnay be preferable to avoid targeting. With tbe cytokines, for example, the role of particular molecules in the regulation of various cellular systerns is well known in the art. In the l r ' '' system, for example,the l r ' " colony-stimulating factors and ' ' regulate the production and function of rnature blood-forming cclls. L~ h~.~ ~ are dependent upon a number of cytokines for ~I~,Iif.laliùll. For example, cytotoxic T ly , ' ~ (CTLs) are dependent on helper T (TH) cell-derived cytokines, such as 1~2, for growth and ll~vlif~,lati~ in response to foreign antig~ns. (7 ' ~ ' arld Doherty, Adv.
Immunol. 27:51, 1979; Male et al., Advanced T ' "~, Chap. 7, Gower Publ., London, 1987; Jacobson et al., J. lmmunol. 133:754, 1984). IL-2, for example, is a potent mitogerl for CTLs (Gillis and Smith, Nature 268:154, 1977), and the of antigen and IL-2 cause 1~ of primary CD4+ T cells in vitro.
The importance of IL-2 for the growth and of the CD8+ CTL in vivo has been ~' ' in models of adoptive ' .,~ in which the therapeutic efficacy of transferred anti-retroviral CD8+ cells is ennanced on subsequent : ' of IL 2 (Cheever et al., J. Exp. Med. 155:968, 1982; Redd~hase et al., J. Virol. 61:3102,1987)~ IL~ and IL-7 are also capable of stimulating the ~li.~..dliUIl of mature CD8+ CTL (Alderson et al., J. Exp. Med. 172:577, 1990). In the case of IL-2, the IL,2 receptors are expressed on T-cells, B-cells, natural killer cells, glioma cells and cells of the monocyte lineage (Smith, Science 240:1169, 1988).
However, the greatest levd of high affinity IL-2 receptor expression is observed in activated T-cells (W411.,~, Ann. Rev. Biochem. 58:87~, 1989). The IL-2 receptor complex consists of three protein r ', a low affinity receptor, ct, Tac or pS5 (Leonardetal.,Nature311:626,1984),an " affinityreceptor,,Borp70 ~II4td~4~a, Science 244:551, 1989), and a sigral r ' protein, y or p64, which interacts with the p70 receptor subunit (Takeshita et al., Science 257:379, 1992).
The ' of the cY and ~ subunits together make up a high affinity foml of the wo ss/2s4s4 2 1 8 7 8 ~ 8 r~l,u. . l~4738 IL-2 receptor (Hatakeyama, Science 244:551, 1989); c~ UUIIJ ' appear tohave the highest affinity (Asa et al., P.N.A.S. 90:41274131, 1993).
Thus, for example, a GDFP including IL-2 can be used to target gene delivery specifically to activated T lylll~hO~ which express high levels of ~ high affinity 5 receptors. The cellular targets of a large number of the other cytokines are known and described in the reviews and other references cited above. r...lh..,.,...~, following the approaches described in those references, any particular cell population or sub-population can be readily assayed for sensitivity to a given cytokine.
The choice of a particular ligand may also be influenced by other activities that 10 may be possessed by the ligand (besides binding to the cell surface). For example, GDrPs having B/T ., derived from cytokines may possess cytokine effector activity tbat can be used to modulate the targeted cells in accordance with the activity of the cytokine. Typically, GDFPs of this type will be prepared by ;"- ~ the entire cytokine coding sequence into a pvl~ ' ' encoding the GDFP; although it 15 will also be possible to remove portiorls of the cytokine sequence which are neither required for binding to the receptor nor essential for cytokine effector activity. In such cases, the GDFPs can provide a ~ ' of activities . , _ (i) binding to specific target cells; (ii) delivery of targeted nucleic acid into the targeted cells; and (iii) cytokirle modulation of the cdls thus targeted. Such a, ' of activities0 will allow, for example, the i ' of particular cells to be coupled to the of the transduced cells. This will be generally ~1~ _ in the context of gene delivery since it can be used to promote the ~,., ' ~ of the targeted cells in a given cell population; and will be ~Li~ a.l.. _ for in vivo gene delivery where it may be otherwise ~., ' ' or impossible to induce the targeted 25 cells to divide, which may be necessary for efficient stable; - ~ of the transferred gene.
In some cases, it will be preferable to make use of the receptor binding potential of a ligand such as a cytokine without ~ effector activity. This may be the case, for example, when a cytokine with suitable receptor binding properties has a 30 negative or unwanted effect on target cell activity. GDrPs of this type can be prepared, for example, from cytokine sequences in which the domain responsible for effector activity has been "~ altered by, e.g., ' insertion or WO 95/28494 2 ~ ~ 7 ~ r~o.. /c 1738 .
-2~
deletion. For example, IL-2 has been subjected to deletion analysis to identify which portions of the sequence are involved in receptor binding and which are critical for cytokine effector activity; see, e.g., F~ B.J. et al., J. Biol. Chem.
262:12306-308, 1987; Brandhuber, B.J. et al., Science 238:1707-09, 1987; Zurawski, S.M. et al., EMBO J. 7:1061-69, 1988; and Arai, K., et al., Annu. Rev. Biochem.
59:783-836, 1990. The receptor binding and effector domains of a rlumber of other cytokines have similarly been ~ see Arai et al., id, and otner reviews and references cited therein.
The rapidity with which novel ligands and their cognate reoeptors have recently 10 been ~l~ul.l.ly cloned has generated a wide a~ay of these molecules. In particular, the ' of direct cDNA expression cloning and screening assays for either induction of ~l~ulircl~Liun of binding to specific cell surface reoeptors on target oells has led to many new molecules being cloned (see, for exsmple, Cosman et al., Trends Biochem Sci 15: 265-269, 1990). The advent of these i ' ' ,, will ~
lead to the cloning of more ligands, includulg cytokines and other proteins that bind to oells which, on the basis of their binding ~l ", r ;~ and specificity may be used in the context of the present invention as the B/T component of tne GDFP. B/T
T - derived from the flk-2/flt-3 ligand (Lyman et al., Cell 75:1157-1167, 1993) will be of interest becsuse the cytokine binds specifically to a receptor, flk-2/flt-3, which is expressed on egrly 1 , cells (Matthews, W. et al., Cell 65:1143, 1991; and Smsll et 81., P.N.A.S. 91:459-463, 1994). In the context of the present invention, GDFPs comprising a B/T component derived from the flk-2 ligsnd could thus be used to direct gene delivery to 1~ . ' ' . stem cells.
While the foregoing principles have been illustrsted using cytokines ss a convenient exsmple, these principles sre also applicable to other ligands cspable of binding to cell surfaces, including for exsmple, sntibodies, lectins, viral binding proteins, cellulsr adhesion molecules, and sny other proteins that sssociate witb cellular surfaoes.
For example, a large number of antibodies to oell surfæ sntigens have been identified snd described. Antibodies to leukocytes have been well ~ l and classified ss the "CDa series of antigens; see, e.g., Coligsn, J. et al. (ed.), aCurrent Protocols in 1' 'lOY," Current Protocols, 1992, 1994. Moreover, techniques for .. . . . . . . _ .. . _ , _ _ _ wossl2s4s4 ~18 781~ r~J~ 5'01738 the isolation of new antibodies specific for a particular target cell are routine in thè art.
Useful antibodies will be those which interact with antigens on the surface of the desired target cells. Antibody/antigen binding can be readily determined and monitored by flow cytometry or other ;,.,.,. ,,.. l.... _l detection methods.
Of particular interest are antigens that are exclusively or preferentially expressed on the surface of particular target cells. For example, the CD34 antigen is expressed on human 1.~ .' ' , - stem cells (Andrews et al., Blood 80:1693-1701, 1992).
T ~ (see, e.g., Zenke, M. et al., P.N.A.S. 87:36S5-3659, 1990), can also be used as a B/T component in the context of the present invention.
Targeting to certain cells, for example respiratory epithelial cells, can also take place via "' ' (Ig) receptors (see, e.g., Ferkol, T., J. Clin. Invest.
92:2394-2400 (1993).
The GDFPs of the present invention can also be chemically modified, for example by the addition of lactose to target the GDFP to ~ U,UIUL ill receptors and thus to I r ' .~a of the liver (see, e.g., Neda, H. et al., J. Biochem. 266:14143-14146, 1991).
Another group of proteins from which the B/ T ~ ~ can be derived are lectins. A number of such molecules, and their cognate receptOrs, have been identifled and ~ l,--, ;, l (see, e.g., the review by Lis ~ Sharon, Ann. Rev. Biochem. 55:35-67, 1986; and l ' cited therein).
Proteins capable of targeting the GDD and thus the GDFP/tNA complex to cell surfaces can also be derived from viruses. Many such viral proteins capable of binding to cells have been identified, including, for example, the well-known envelope ("env") proteins of l~uv~h~ a~ ' - proteins of RNA viruses such as the influenza virus; spike proteins of viruses such as the Semliki For~st virus (Kielian and wulh (1990) Mol. Biol. Med. 7:17-31~; and proteins from nu ~ viruses such as ad.~ùvu~_a (see, e.g., Wickham et al., Cell 73:309-319, 1993).
As an illustrative example, in the murine leukemia virus (MuLV) system, it is well known that the . ' region of the gp70 molecule is involved in binding to cell surface reCeptorS, see, e.g., Heard and Danos, J. Virol. 65: 4026-4032, 1991.
Battini et al., J. Virol. 66: 1468-1475 (1992) have also reported that portions of the W095128494 21~?8~8 r~ mscl738 amino-terminal region of gp70 can be exchanged in order to switch binding to different MuLV env receptors without interfering vith the ability of the protein to interact with plSE TM protein (and, thereby, to mediate virai uptake); see also Weiss, R. et al. in Weiss, R. et al. (eds.), RNA Tutnor Viruses, Cold Spring Harbor, New York (1984 and 1985). Similarly, in the human ' ~ .y virus (HIV) system, mutational anaiysis of gpl20 has identifled portions of the molecule which are critical for binding to the CD4 receptor, see, e.g., Kowalski, M. et ai., Science 237:1351-1355, 1987.
Yet another approach to identify a region critical for receptor binding is as follows: an antibody known to inhibit binding can be used to immuno-affinity purify a cleavage fragment of the viral binding protein; which fragment is then partially sequenced to identify the ~ull~ r ' domain of the viral binding protein, see, e.g., Lasi~ey, L.A.
et ai., Cell 50:975-985, 1987. Such techrliques can be employed in the present invention to generate GDFPs in which the M-D component remains capable of mediating uptake of the GDFP/ti~A comple~ (as describe~i below), but the specificity of bindirlg is principally determined by the presence of, e.g., cognate cytoicine receptors ' ~ to a portion of the B/T, , t, rather than virai bindmg protein receptors.
Another illustrative example of a virai protein that can be used is the G protein of VSV, which has been utiiized to target infection by retrovirai vectors; see, e.g., Emi et al., J. Virol., 65:1202-1207, 1991.
Another group of proteins from which the B/T --r ' can h derived are celluiar adhesion molecules. A number of such molecules, and their cognate receptors, have been identified and, 1 "". .; 1 (see, e.g., Springer, T., Nature 346:425-434, 1990, and ~ cited therein).
(2) Memllr~nP-~ (M-D) ('nmnnn.~n~c r~ ~ disrupting, . are protein sequences capable of locaily disrupting cellular membranes such that the GDFP/tNA complex can traverse a cellular membrane.
M-D -----r ' faciiitating uptake of the GDFP-targeted nucleic acid complex by target cells are typicaily ' -active regions of protein structure having a WO9S/28494 21 8 7 ~ u..,''r4738 ll~d~uyhulJ;c character. Such regions are typical in membrane-active proteins invoived in facilitating cellular entr,v of proteins or particles.
For example, viruses commonly enter cells by Cllduu.~ ~ua;a and have evolved for disrupting endosomal m~mhrAnPc Many enveloped viruses encode 5 surface proteins capable of disrupting cellular mPmhrAnPc including, for example, ICLlUVUh~ , influenza virus, Sindbis virus, Semliki Forest virus, Vesicular Stomatitis Virus, Sendai virus, Vaccinia virus, and mouse hepatitis virus; see e.g., Kielian and Juub_.wuil~, Mol. Biol. Med., 7:17-31, 1990; and Marsh & Helenius, Adv. Virus Res., 36:107-151, 1989. The mechanism fûr viral entry, m which a Yiral binding 10 protein binds to a specific cell surface receptor and ' . ~ mediates virus entry, frequently by means of a h~luyhul~ disruptive domam, is a eommon theme among enveloped viruses, including influenza virus, and many sueh moleeules are known to those skilled in the art, æe, e.g., Hunter ar~d Swanstrom, Curr. Top.
Micro. and Immunol. 157:187, 1990; and the review by White, J., Seienee 258:917-924, 1992; and ~ ' ' - reviewed therein.
By way of illustration, the M-D ~ A ' of the preænt invention ean thus be derived from a portion of a viral binding protein that is normally involved in mediatmg uptake of the virus into a host eell, or a mutein of sueh a portion of a bindmg protein.
The portion of the GDFP that may be derived from sueh a viral bmding protein may, 20 but need not, also eontam the portion of the bindmg prooein that eauses the viral partiele to associate with a specifie receptor on a target eell (whieh latter portion may thus funetion as a B/T rnmrr,n,~nt as described above). A large numbcr of viruses have been ~ ; 1 and, for many of these, the nueleotide sequence of the viral - genome has been published~ The binding proteins eneoded by various viruses generally 25 share functional homology, even though there may be ' ' ' variation among theprimary amino acid sequenees. Using the IdlUVUI..~ to illustrate, the native env gene product is typieally a polyprotein precursor that is ylutuvl.~ui~lly eleaved during transport to the cell surfaee to yield two l,vlv~ . a bl~uay ' y~ l., on th-e external surfaee (the "SU" protem) and a ll.~ spamling or i 30 protein (the "TM" protein); see, e.g., Hunter, E. and R. Swanstrom, Curr. Topies Microbiol. Immumol. 157:187-253, 1990. The SU proteins are resporlsible for binding to speeific receptors on the surface of target eells as a ftrst step in the infection W0 95/28494 2 1 8 7 8 1 8 ~ 738 process. The TM proteins, as well as associating with viral core proteins through their C-terminal ends, are responsible for a critical membrane fusion event which takes place after binding and allows entry of the virus irlto the cell (See, e.g., Hunter and Swanstrom (1990) Curr. Top. Micro. and Immumol. 157:187; Kielian and ~i _ W;lLI.(1990) Mol. Biol. Med. 7:17-31; and Marsh ~ Helenius (1989) Adv. Virus Res., 36:107-151). The membrane fusion event is ~ r' ' ' by a ~y~u~ ub;~
pul,Y~ id~ sequence present at the amino terminus of the TM protein. Examples ofthese pairs of SU and TM proteins and the viruses which produce them are: gp52 and gp36 from mouse mammary tumor virus (Racevskis, J. et al., J. Virol. 35:93748, 1980); gp85 and gp37 from Rous sarcoma virus (Hunter et al., J. Virol. 46:920, 1983);
gp70 and pl5E from Moloney murine leukemia virus (Koch et al., 49:828, 1984); gp70 and gp20 from Mason Pf~er monkey virus (Bradac, J. et al., Virology 150:491-502,1986); gpl20 and gp41 from human ' - ,Y virus (Rowalski, M. et al., Science 237:1351-1355, 1987); and gp46 and gp21 from human T-Cell leukemia virus(Seiki et al., Proc. Natl. Acad. Sci. 80:3618, 1983); and others described in the references cited herein. The functional similarity among these types of proteins, is further illustrated by the well :' ' I' f "~ u,luL~r _ " in which the core proteins and nucleic acid are provided by a first virus and the envelope proteins (~' _ host range) are provided by a different virus (see, e.g., Vile et al., Virology 180:420, 1991; Miller et al, J. Virol. 65:2220, 1991; and Landau et al., J.
Virol. 65:162, 1991). E~amples of I~UVII~ J which can be used to derive fragments for use in the present invention include murine letIUV;~D such as Harvey murine sarcoma virus (Ha-MSV~, Kirsten murine sarcorna virus (Ki-MSV), Moloney murine sarcoma virus (Mo-MSV), various murine leukemia viruses (MuLV), mouse mammary tumor virus (M~IV), murine sarcoma virus (MSV) and rat sarcorna virus (RaSV);
bovme leukemia virus (PLV); feline le~lUV;IU~J such as feline leukemia virus (FeLV) amd feline sarcoma virus (FeSV); primate l~UUV;IUD_J such as baboon ~ l ,rD. , - virus (BaEV), humarl ' ,Y viruses (EIIV-I and HIV-II), human T-cell leukemia viruses (HTLV-I and HTLV-II), Gibbon ape leukemia virus, Mason Pfr~er monkey virus (M-PMV), simian ' - ~ virus (SIV) and simian sarcoma virus (SSV); various l~ iYUl~.J, and avian l~uvilh~D such as avian ~Iyi' ubl~Dtu~;D virus, avian leukosis virus (ALV), avian IllJ~lolJl~DluaiD virus, avian sarcoma virus (ASV), ., ., . , , .. , . , _, . , .. , ., .. ,, . _ . , ,, .,, .. ,,,, , .,,,,, ,,, . ,, _ , . , _ _ _ , _ = . , . =

W095/28494 2 1 87 8 1 8 P~ t738 avian .cl; l-,. .,.l,.~h. ~ associaoed virus (REV-A), Fujinami sarcoma virus (FuSV), spleen necrosis virus (SNV) and Rous sarcoma virus (RSV). Many other suitable lcnuvilhD~D are known to those skilled in the art and a taxonomy of ICLiUViUU~.,s is provided by Teich, pp. 1-16 in Weiss et al., eds, RNA Tumor Viruses, 2d ed., Vol.2, 5 Cold Spring Harbor, New York. Plasmids containing retroviral genomes are also widely available from the ATCC and other sources.
Infectious virions have also been produced when non-retroviral binding proteins,such as the G protein of vesicular stomatitis virus or the l __' of influenza virus, have been pseudo-typed onto retrovirus cores (see, e.g., Emi et al., J. Virol.
65:1207, 1991; and Dong et al., J. Virol. 66:7374, 1992). These latter examples indicate that there are functional ' between various viruses and their mode of entry mto cells which will allow the use of viral binding proteins from a variety of sources. Influenza I __' has also been reported to enhance the uptake of poly-L-lysine-based chemical conjugates (Wagner et al., P.N.A.S. 89:7934-7938, 1992).The sequences of a large number of viral binding proteins are known, and are generally available from sequence databases such as GENBANK. r;...lh. .,- "~
p~l~lll~l~..Jlid~.D encoding viral binding proteins can be readily obtained from viral particles themselves. Also, since many different genes encodmg viral binding proteins h~ave been cloned and . ~ . .; i. plasmids containmg DNA encodmg the binding 20 proteins are available from a numh~er of different sources. r~ iduD encoding viral binding proteins can also be obtamed using p~ l.,la:~C chain reaction (PCR) ' ' ' 0~, as described, for example, by Mullis and Faloona (1987) Meth.
155:335.
As an illustrative ~ ' ' of the present invention, a GDFP may comprise a 25 region of a gene encoding a viral binding protein including a B/T . t, in which case the GDFP cam be used to target cells including those normally susceptible to the virus from which the gene was derived. In other, ~ ' of the present mvention, the targeting may be restricted to cells h~earing receptors for other types of ligands, discussd above umder the description of the BIT: . For example, 30 where an M-D component is derived from a viral binding protein that retains the ability to bind to the viral receptor, but it is desirable to limit targeting to cells bearmg, e.g., an appropriaoe cytokine receptor, there are several approaches that can be used to W095/28494 21 87~18 r~ .ol738 .

achieve such specificity. One approach is to utilize a BIT compoDent which is based on a cytokine with a very high binding affinity for the desired target cells compared to the bir~ding affinity of a domain derived from a viral binding protein for the native viral binding protein receptors. Since many of the cytokines are known to exhibit very high affmity binding to their receptors, and since it will be feasible, for example, to base the M-D component on a lower-binding-affinity viral binding protein, targeting can be effectively focused upon those cells bearing a cognate cytokine receptor. Another suitable approach to limiting binding is to derive the M-D coinponent from a mutant viral binding protein in which the mutation has disrupted the abiliq of the protein to engage in binding via the native viral binding protein receptor but has not interfered with the ability of the viral binding protem to mediate viral uptake. Plasmids encoding such mutant viral binding proteins are available in the art; and it will also be well within the ability of one skilled in the art to prepare new versions of such viral binding protein mutants by deleting portions of the coding region or by i . ' amino acid ' mto the coding sequence as described above.
While the foregoirlg principles have been illustrated using viral proteins as a convenient example, tbese pririciples are also app~icable to other pG~Li~.. capable of disruptmg oellular membranes (see, e.g., the review by White, J., Scienoe 258:917-924, 1992, and I ' reviewed therein).
Other domains that are ~ "y and/or structurally analogous c;m be derived from various vit~l, prol~uyotic or eukaryotic sources. As a further specific example, bacterial toxins such as diphtheria toxin have a specific domain with a highly ~Ip~ Llh,.ll structure and a hJL, A ~ ~ character (known as the ~TM~ domain in tbe case of diphtheria to~in) tb~t becomes protonated at low pH and disrupts cellular rn~n~ C. facilitating erltry of the toxin into the oells (see, e.g., Choe et al. (1992) Nature 357.216-222; V7nA~r~ et al, J. Biol Chem 268: 12077-12082, 1993; amd Parker & Pattus, Trends Biochetn. Sci. 18:391-395, 1993). Toxirls such as r~ ~1 exotoxin A have a similar membrane-disrupting domaitl (see, e.g., Strom et al., Ann. N.Y. Acad. Sci. 636:233-250, 1991). Similar M-D ~ , can be derived from other bacterial toxins such as hemolysin (Suttorp et al., J. Exp. Med., 178:337-341, 1993). As described herein, inclusion of such membrane disruptive 2i8781~
wo gsl28494 -31- r~l,. c~ .738 ..,.,l,.. ~ in the GDFP would facilitate membrane disruption and entry of the GDFP-tNA complex into the target cells.
Cytolytic pore-forming prooeins, such as ~ Lul~ul O, perforins expressed by cytotoxic T 1.~ , and S.aureus alpha toxin also have the ability to disrupt .,.. ~,.. (see, e.g., Ojcius and Young, Trends Biochem. Sci., 16:225-230, 1991;
Suttorp et al., J. Exp. Med., 178:337-341, 1993). Streptolysin O has been shown to facilitate uptake of DNA by cultured cells when added to the culture medium (Barry et al., r - ~ . 15:1018-1020). There are many bacoerial cytolysins which have the capability to induce membrane disruption (see, e.g., Braun and Focareta., Crit. Rev.
Microbiol. 18:115-158, 1991; and van der Goot et al., Nature 354:408-411, 1991).Membrane disruption often occurs by means of a pH-induced h~J.u~h~;~ change in the prooein, but this can also occur by enzymatic means, such as those involving(see, e.g, Braun and Focareta., Crit. Rev. Microbiol. 18:115-158, 1991 (and references cioed therein); and London, Mol. Microbiol. 6:3277-3282, 1992).
IS Where a pH shift is required to induce the membrane disruption function, there are several ways in which this can be achieved. For example, the GDFP/tNA complex may be taken up through acid, l..~.. -. or the pH of the, " ' medium may be transiently lowered to mediaoe activation of the membrane disruption function. In some cases (diphtheria toxin for example), enzymatic nicking of the membrane active component prior to an induced pH change in the , ' medium is believed to promooe membrane disruption (see, e.g., Sandvig and Olsnes, J. Cell Biol. 87:828-832, 1980; Moskaug et al., J. Biol. Chem. 263:2S18-2525, 1988; and Zalman and Wisnieski, Proc. Natl. Acad. Sci. 81:3341-3345, 1984). Well-known enzymes such as trypsin and urokinase have been r ~ used to provide the nicking activity in vitro (see, e.g., Williams, D.P., et al., J. Biol. Chem. 265:20673-20677, 1990).Enzymes capable of providing the nicking activity are also known to be found on cellular surfaces (see, e.g., Williams, D.P., et al., id.). Exemplary r~ and ;.... of GDFPs containing the diptheria toxin i ' region are described below in Example 8.
Other sources of M-D - . include bacterial prooeins that promooe entry of organisms into cells, such as the 52kD entry prooein of ~ ~LubuLt~ . L~.lL.
(Arruda et al., Science 261:1454-1457, 1993); the inoernalin prooein of L~steria WO 95128494 . ~ ~I/.J.,. _.'01738 .

monocytogenes (Portnoy et al., Inf. Imm. (U.S.) 60:1263-1267); and the invasin protein of Yersinia enterocolitica (Young et al., J. Cell Bio., 116, 197-207), among others.
Synthetic analogs of lll~,ll.Jl~ disrupting domains can also be made. See, e.g.,Kaiser and Kezdy, Science 223:249-255, 1984.
~3) Trarlsport/L~roli7~rirln (T/L) Com~
Transport/lnr~li7~tirn ~ mediate or augment the transport and/or Irr~1i7~tir,n of the GDFP/tNA complex to a particular sub-cellular ~ such as the nucleus or ~
A number of sequences that mediate transport and/or ir~r lli7~tirn of proteins have been identified. These include, by way of the nuclear irr~l ' sequence (nls) of SV40 T antigen (Colledge, el ai., Mol. Cell Bio. 6:4136-4139, 1986);
and the I~IV matrix protein (Bukrinsky et al., Nature 365:666-669, 1993). These are typically short basic peptide sequences, and may aiso be bipartite basic sequences (see, e.g., Garcia-Bustos et al., Biochim. Biophys. Acta 1071:83-101, 1991; and Robbins et al., Cell 64:615-623, 1991). Nuclear lr,r~li7~rir,n sequences have been fused toL~vlv2~ proteins and shown to confer on them the property of nuclear Ir,r~li7~tirln (see, e.g., Biocca et al., EMBO J. 9:101-108, l99v). In the case of the human esv - ogen receptors, for example, fusion proteins traffic to the nucleus in an estrogen-dependent fashion (Ishibashi et ai., J. Biol. Chem. 269:7645-7650, 1994). These sequences can be readily ~ ,ull ' into the GDD by ' DNA, - :I--vl~
to facilitate nuclear Irr~1i7itinn of the desired GDFP/tNA complex. GAL4 has also been shown to possess nuclear Irr~1i7~tir~n properties in yeast (see, e.g., Silver, et al., P.N.A.S. 81:5951-5955, 1984), and thus, as a component of a GDFP, GAL4 may be used as both an NBD and a GDD with a role in transport/lr,r~li7~ti~1n (4) ReDlicon Inte~ration (Rl) ~'r,mnr,nrntc Replicon integration ~ mediate or augment integration of the targeted nucleic acid into a replicon of the target cell, such as a ~hlu~v~u~ ,. In many instances in gene transfer and gene therapy it is ddv~i..6_v~ to obtain stable integration of transferred DNA into the genome oi- the target cell. The GDFP can W095128494 P~l/u.. '.'04738 facilitate such integration. Also, as described herein, a particular advantage of the Type I GDFP approach is that it not only allows the ~l~.;, l,;.,.,.. :.i. attachment of delivery ~ to the tNA, but also allows the GDFP to be positioned at pre-determined locations with respect to the tNA. In the case of a replicon integration 5 component such as an integrase, the GDFP can be positioned in proximity to terminal integrase recogtution sequences as a means of facilitating int~gr:~rit~n as described in more detail below.
DNA-protem interactions can mediate integration of DNA into the rr~n~ n genome. For example, the iMegration of all known IC..IUVUL..~ takes place in an lû enzymatic reaction that makes an ~ ,.. cleavage of the host DNA and ligates the reverse-transcribed retroviral genome to the free ends of the host cell DNA. This reaction is mediated by the retroviral iMegrase (or ~IN~) protein, and it is well known that the IN protein interacts with a minimal r~nbcr of bases present on the ends of the pre-integrative viral geDome to achieve inr~ ri~ln Indeed, DNA sequences bearing15 the IN sequence recogrution motif can bc inserted into free DNA m vitro by purified IN prooeins (see, e.g., Bushman et al., Science 249:1555-1558, 1990; and Katz et al., Cell 63:87-95, 1990; see also, Brown et al. Cell 49:347-356, 1987; and Roth et al., Cell 58:47-54, 1989). For example, the MLV, HIV and RSV IN proteins are each known to mteract with a distinct short IN sequence O motif present at each 20 end of the linear pre-- ~ .~. viral DNA substrate to mediate its mtegration into the host cell replicon. In vitro integration mediated by purified IN protein has been ' usmg either free ~ t - ' or synthetic DNA substrates bearing the IN lc, O scquence motif (see, e.g., Katz et al., supra; amd Bushman and Craigie,J. Virol. 64: 5648, 1990). Synthetic DNA substrates can be rcadily engineered by25 inserting a unique rcstriction enzyme site (typically Nder), flanked by the ~IN O sequences, into a plasrnid vector. Digestion of the vector with Ndel yields a DNA substrate with 3' rccessed ends preceded by the highly conserved 5'CA-OH ,' ' ' and the rcmainder of the appropriate IN recoglution motif, which resembles the processed ends of the prc- ~ .~, viral DNA with which IN
30 interacts to mediate intcgration. This approach has been ~ "~ used to in vitro iMegration of such lincarized DNA substrates mto double-stranded DNA in vitro by purified avian retrovirus IN (Kae et al., supra), 2t~78t8 WO 95/28494 ~ /L,.._.'C ~738 MoMLV IN (Bushman and Craigie, J. Virol., supra) and HIV IN (Bushman et al., Science, supra). The IN recognition sequences used on the termini of the subsv~ate DNA in these ~ were short (10-30 base pairs), .1 ",. ~ that uloljuu~ DNA substrates with short terminal IN sequence rerr~n~ n motifs can be 5 integrated into double-stranded DNA by IN. Moreover, these ~ document successful integration of both ends of the DNA substrate into the target DNA, asopposed to the ~ ' ' integration I~ which assay only for integration of a single end of the substrate DNA. These e-l . ;.. t~ document that linear DNA
substrates bearing short tcrminal IN recoglution motifs can be integrated into double-sv~anded DNA in vitro by purified IN protein. Thus, the foregoing provide further evidence of the utiliq of the present invention, in that IN .
can be included in the GDFP and can act in concert with termir~l IN ,, sequence motifs present OD the (substrate) vNA to mediate efficient integration.P fusions bet veen integrase and h~vlv~ ~ prooeins have previously been c-~nC~n~rt~l expressed and shown to retain integrase enzymatic activity (see, e.g., Vink et al., J. Virol., 68:1468-1474, 1994). Moreover, Bushman (PNAS 91:9233, 1994) has shown that ' fusions can be made between integrase and a sequence-specific DNA binding protein, and that such rusions retain integrase activity and sequenoe-specific DNA binding.
Thus, for example, by including an Rl component derived from an integrase protein in the GDD, and using a tNA bearing the IN recoglution sites, tbe GDFP can be co ' ~ with the targeted nucleic acid (tNA) bearing the integration rerogr;rirn motif and thus achieve integration of the tNA into a replicon of the target cell. This system would also allow. in , with an appropriate binding domain in the NBD, for the association of the Rl component with the free ends of the tNA.
This would be ad~ u~ since the IN proteins of l~tlUvul~_~ runction to rnediate integration at the rree ends of pre- ~, viral DNA. In the present invention, this can be achieved by utilizing a Type-I GDFP in ~ , with a linearized tNA
containing the cognate recoglution sequence (~CRS~) for the NBD at (or in close proximity to) the ends of the tNA bearing the terminal IN sequence recognition motif (preferably less than 500 nucleotides from tne IN sequenoe, more preferably less than 200 ' ' . most preferably less than 5û n~lrl~Oti'l~C~). To generate the tNA for wo gs/28494 2 1 8 7 8 1 ~ r~"~)~ 5~04738 *

gene delivery, the tNA can be consttucted, for example, with a unique NdeI site between the two IN recognition motifs, flanked by the cognate rerogn~ n sequence of the NBD. Digestion with Ndel would tnen generate a linear DNA molecule with 3' recessed ends preceded by the 5'CA-OH ~ the remainder of the IN
Sequence recognition motif, and the CRS for the NBD, m t}tat order. In tnis way, the terminal IN recognition sequences would be closely linked to the cognate ~, sequence for the NBD. Typically, the Ndel site/IN lc O sequence/CRS cassette would be inserted into the plasmid backbone of the vector. However, it is possible to construct a tNA devoid of any extraneous or l ' ' ' sequences; for example, a tNA devoid of any bacterial plasmid sequence can be generated by flarlking each end of the " expression cassette in the tNA with a CRS, followed by one half of the IN recognition sequence, followed by an Ndel site. Digestion by Ndel would then generate a linear tNA DNA fragment, which could be readily purified from the plasmid backbone fragment, havmg on each end the IN ~, sequence and the CRS.
Removal of plasmid backbone sequetlces may be desirable to achieve optimal gene rcgulation in the transduced cells. Bindit~g of the GDFP would then locate the Rl component containing the IN region m close proximiq to tne sites on the tNA withwhich it can mediate efficient integration. An analogous stratcgy can bc used with the AAV Rep protein and viral l-lRs (see, e.g., Owens et al., J. Virol. 67:997-1005 (1993), and the review by Caroer, B.J., Current Opinion Biotech. 3:533-539 (1992) and y..~ . - reviewed therein). ~ " other systems such as the Pl cre ' . the yeast FLP l. ' , the yeast SRI-dcrived R ' amd the Tyl inoegrase (see, e.g., Kilby et al., Trend Genet., 9:413-421, 1993; Moore amd Garfinkel, PNAS 91:1843-1847, 1994) can be used in the context of 25 the present invention using fusions with appropriaoe NBDs, cis-acting .
recogtution sequences and CRS elements, in an analogous fashion to that described above for the moegrase fusions.
the Rl domain may be required for optimizing irlteO~ration activiq, especially in situations m which the protein from which the Rl domain is 30 derived functiotls in multimeric form. Thus, for example, many native retroviral IN
proteins are dimeric or multimeric in form (see, e.g., Jones et al., J. Biol Chem 23:
16037, 1992; reviewed in Skalka, Gene 135:175, 1993). I~ of the IN

21 87~1 8 w0 95/28494 E~l/ u.. ~ ~738 domam can be CUUY~,II~ILI~Y achieved by, for examp}e: (a) Cu...~lu~ ; tandem repeats of the IN component in the GDFP, preferably separated by a flexon; (b) dimerizing the GDFP by insertion of a protein ~ i.,., motif, e.g., a leucme zipper motif (see, for example, Hu et al., 1990, Science 2~0: 1400); (c) adding free IN protein to an IN-containing GDFP (since IN proteins have a natural tendency to ' ); or (d)the CRS in the tNA such that multiple GDFP molecules bind to each end oF the tNA. Cl'., ', - ;. ,- - - of the above strategies can also be used. This would result in furd~er ' of the IN component and thus a more active integration complex.
Another strategy to acnieve '- - of tne RI domains, and also to achieve more efficient concerted mtegration of the tNA (i.e. having both ends of the tNA mtegrate into the replicon), would be to engineer the system to bring the free ends of the linear tNA together. This can be achieved in a number of ways in the context of the present mvention. First, the GDFP monomers can be designed in such a way that 15 they self-dimerize, using a leucine zipper or other motif as described above. Thus, .l, - ;,- ;.. of tne GDFP bound to the tNA would result in close apposition of the free ends of the tNA, since the CRS is located near these termini. A second approach would be to use a second, separate DNA binding protein with additional cognate recognition sequences preserlt near the termini of the tNA to bring the ends of the tNA
20 together (for this purpose any of the DNA bindmg domains alluded to above could be used m dimeric form together with a tNA having the appropriate cognate recognition sequence to associate the free DNA ends). Such strategies would bring the free ends of the tNA in close apposition to one another and thus may further enhance the frequency of concerted irltegration. Several approaches can be used to avoid amy potential2~ problem that may arise from GDFP/tNA complex ~ j.aLiu~ ~i.e. integration of the ends of the tNA molecule mto itself), or ~ " of one tNA molecule mto another. Although ~ . ' of the GDFP with the tNA could be done at 4C, thus reducing IN enzymatic activiy, placing the complex onto cells at higher ~....l .. r.. -r could lead to such unwanted integration events. A preferred approach is to use a30 conditional Rl moiey. Such conditional Rl moieties can be dependent on chemical or proteun co-factors, or can be mut;mts that are conditional for full activiy dependent on or other variables, such as the presence or absence of inhibitors or co-.. ... . . _ . . ... . .. . . . . . _ _ _ . _ . _ _ _ _ _ WO 95128494 2 l 8 7 8 1 ~ r~u~ ~4738 .

factors. For example, in the case of IN, t~ l,.u.~-sensitive (ts) IN mutants bave been made that are active oniy at certain t~l.ly~ (see, for exampie, S~ iJ~
et al., Virology 192:673, 1993). Tlqe use of a ts RI component would allow exposure to and uptaice of the complex by cells to be done at the non-permissive ~ r (such that the Rl component would not be active), followed by switching to the permissive ~ , c once the complex was taicen up into the nucleus, allowing the RI component to be active in the context of the host cell replicon and thus accomplish tbe desired integration.
Thus, inclusion of an Rl component in tbe GDFP can be used to enhance frequencies of integration. The GDD can consist of an RI component alone, or it can in addition comprise one or more of the other ~ discussed above. Where tbe RI component is ti~e sole component in tile GDD, the NBD would function to associate tbe RI component more stably andlor more specificaily with tile free ends of the ti~A
than is possible througiq, for example, use of the ' native IN protein alone.
By virtue of the NBD bindimg, the RI moiety is more tightiy associated with tile WA
termini during tiqe i ~ process and can mediate integration into the host cell replicon. Where the RI component is the sole component m the GDD, the tNA/GDFP
complex can be delivered by any of the standard means of l, ~ F I ;~ , such as li r rl. . ~ etc., and the tesulting cells would have an eniqanced frequency of stable gene delivery as a . of enhanced irltegration of the tNA
Alternatively, the complex can h delivered by other non-virai means, including for example the use of ~If ' ' systems such as viral capsid proteins. r evidence has confirmed that virai capsid proteins can be used to introduce DNA into " ceiis (see, e.g., Forstova et ai., Hum. Gene Ther., 6:297-306, 199S).
In certain cases, such as the retrovirai IN or AAV Rep proteins, '" 'l""" --`` of the GDD can aiso function as effective NBD ~ , and thus fuifiil a duai function in the GDFP by virtne of tiqeir abiiiq to bmd nucleic acid (see, e.g., i'~rongstad ~c Champoux, J. Virol. 64:2796-2801, 1990; Owens et ai., J. Virol. 67:997-1005 (1993); and the review by Carter, B.J., Current Opinion Biotech. 3:533-539 30 (1992) and l~I.I;r_l;. reviewed therein).

21~78t~
wo 95/28494 P~l/u~ o 1738 .

2. The Tar~eted nllrlPir ;~ri~i (tNA) The targeted nucleic acid (tNA) is a poly~l,..l~viide, or analog tiDereof, to bedelivered to a target cell. Thus, targeted nucleic acids include, for example, ol ~ ' ' and longer polymers of DNA, RNA or analogs thereof, in double-5 stranded or single-stranded form. The tNA may be eitber circular, supercoiled or linear. A preferred exarnple of a targeted nucleic acid is a DNA expression vector comprismg a gene (or genes) of interest operably linked to a h.~ ' control region (or regions) and a cognate recognition sequence capable of being bound by the NBD domam of the GDFP. The i ~ ' control region may be selected so as to 10 be specifically activated in tbe desired target c~lls, or to be responsive to specific cellular or otber stimuli.
Targeted nucleic acids may also include, for example, positive and/or negative selectable markers; thereby allowmg the selection for andlor against cells stably expressimg the selectable marker, either im vih~o or in vivo.
Use of the present mvention to deliver RNA would enable the i~, ' of RNA decoys (Sullenger et al., Cell 63:601-608, 1990); ribozymes (Young et al., Cell 67:1007-1019, 1991); and antisense rlucleic acids (Vickers et al., Nucleic Acid Res., 19:3359-3368), for example.
In Type-l GDFPs, the targeted r~uckic acids are recognized and bound by the 20 GDFP by virtue of specific cognate ~ , sequences to which the rlucleic acid bindmg domain (NBD) of the Type-l GDFP binds. Both DNA and RNA binding domains have been isolated from proteins tbat bind to particular nucleic acids in a sequence-specific fashion. Inclusion of such a cognate recoglution sequence im the targeted rlucleic acid allows for specific bindmg of the GDFP to the tNA. P ~, 25 sites for rnany nucleic acid binding proteins have been identified (see, for example, Mitchell & Tjian, 1989; and other references herein).
Bmdmg of sequence-specific bmding proteins to DNA tends to be more avid when the recognition sequence motif is ' ' (see, e.g., Hochschild and Ptashne, Cell 44:681-687, 1986). Accordingly, the cognate recognition sequences may 30 be ' ' in the targeted rlucleic acids so as to enh~mce the bindmg affinity orselectivity of a GDFP for its cognate tNA. This could also have other advantages, such as increasing the effective amount of the GDFP bound to the tNA, or promotmg W095128494 2 1 8 7 ~ 1 8 1~ "~,,., ~'01738 .

romr~ri(m/~ ' of the tNA by sequence-specific or sequence-non-specific NBD .: ..., .l,..".. ,~
Typically, but not necessarily, the cognate recognition sequences in expression vectors will be piaced in the plasmid backbone of the vector. This also applies to otner cis-acting sequences that are needed in the tNA to facilitate gene deliYery. However, it may be desirable to remove plasmid backbone sequences from tne DNA to be transferred. In this case, the expression cassette can be . ~ .ly flanked by restriction enzyme sites, such that restriction enzyme digestion separates the backbone from the ' expression cassette. The expression cassette can then be purifled away from the plasmid backbone for use in i eAI,, Clearly in this case the CRS would need to be located on the fragment bearing the expression cassette.
It is also possible, of course, to const~uct the GDFP so as to bind to more than one tNA.
As discussed above, the tNA can also be bound to tbe GDFP via . non-specific in addition to sequence-specific In a Type-l GDFP, such sequence-non-specific i ` ~ can be mediated by auxiliary ~
derived from sequence-non-specific binding proteins, as discussed above. Such auxiliary non-specific binding , can also serve to compact or otherwise .~ '1, ~ the targeted nucleic acid; see, supra.
The targeted nucleic acids can also include, for example, non CA~ d DNA, such as sequences l ' _ to sequences present in a target cell replicon, that canthereby mediate ' ' ~, This can be used to facilitate the stable integration of the targeted nucleic acid, or a desired portion thereof, into a specific site in a replicon present in the target cell, such as a specific site in a cellular .
This may be useful, for eAample, to achieve a desired level of expression of the tNA
by integration at a desired ~Ll ' site. TT.. ~ ' can also be used to alter a specific DNA sequence in a target cell replicon (see, e.g., Thomas &
Capecchi, Cell 51:503-512, 1987).
For longer tNA sequences, or where the tNA uptake mechanism (whether part 30 of the GDFP or not) is known or suspected to be sensitive to the size, form or charge of rlucleic acids and/or complexes to be delivered, such as ' mvolving ~io.JtUD;D, it may be desirable to condense and/or charge neutralize the tNA. This 21 878~ 8 WO 95/28494 1 . ~ 738 .

can be achieved by mixing the tNA with any of a number of prooeins or other agents (collectively referred to as "-U~ a~ agents") that can condense and/or charge neutralize nucleic acids. ('~mr~rinV agents include, for example, histones (see, e.g., von Holt, Bioassays 3:120-124, 1986; and Rhodes, Nucleic Acid Res., 6:1805-1816,1979); or pvl~ iid.,;, derived therefrom (Rodriguez et al., Biophys. Chem., 39:145-152, 1991); as well as the non-histone high mobility group proteins. Poly-L-lysme or other polybasic amino-acids can also be used as c~ v agents (see, e.g., Li et al., R;". h~ y, 12:1763-1772, 1973; and Weiskopf and Li, Biu~ 16:669-684, 1977). Similarly, other pOly~,a~iùlli~. polymers such as polyamines, for examplespermine and Sr~mi~in~, and cationic lipid-containing polymers cam also be used to condense and/or charge neutralize nucleic acid (see, e.g., Feuerstem et al., J. Cell.
Biochem., 46:37-47, 1991; and Behr, Bioconj. Chem., 5:382, 1994). Retroviral " 1. r ~ proteins can fulfill a similar role (see, e.g., Gelfand et al. J. Biol. Chem., 268:18450-18456, 1993).
15 Alternatively, . v agents can be i.. ~.ulr ' as an additional component of the GDFP. Also, some sequence specific binding proteins, such as GAL4, which exhibit a range of bindmg affmities to different cognate nucleic acid sequences may also be used in this capacity, and in this regard would function as an NBD with both nucleic acid binding and . . properties.
C~ ~ ~ agents might also be . ' as mediators of indirect bindmg between the tNA and the NBD domain of the GDFP (for example, the NBD domaim can be bound to tbe compacting agent and the ~ . ~ agent bound to the tNA).
AC~ nnh~v of GDFPs Preferably, the GDFP is prepared as a single pc~ i~ fusion protem generated by ' DNA . 1~ To generate such a GDFP, sequences encoding tne desired r of the GDFP are assembled and fragments ligated into an expression vector. Sequences encodmg the various f, .~ may be assembled from other vectors encoding the desired protein sequence, from PCR-generated fragments using cellular or viral nucleic acid as template nucleic acid, or by assembly of synthetic ~ ' encoding the desired sequence. However, all nucleic acid s~quences encodmg such a preferred GDFP should preferably be assembled by in-fMme wo ss/2s4s4 2 1 8 7 8 1 8 P~ 738 fusions of coding sequences. Flexons, described above, can be included be~ween various c.".,l..", .,l~ and domains in order to enhance the ability of the individual f~ ` to adopt ~" 8';L~.U;'~ - relatively; 1 ~.. l .lly of each other.
Although a Type-l GDFP is preferably assembled and expressed as a single 5 ~oly~ ide chain, one or more of its domains or ..~ may be produced as a sepa}ate chain that is ~ , linked to the GDFP by, e.g., disulfide bonds, or chemical ~"'`i 'E,"';"" It is also feasible to prepare complexes in which domains such as the NBD arld the GDD or their ~ are physically associated by other than lC_ ' means, either directly or indirectly, for example, by vir~ue of non-covalent 10 ;, t ~ or via co-ln~ 7~tinn on a ~,., or lipid surface.
The GDFP may be expressed either in vitro, or im a ~ or eukaryotic host oell, amd can be purified to the extent necessary. An alternative to the expression of GDFPs im a host cell is synthesis in vitro. This may be adv ~ in ~- in which high levels of expression of a GDFP might interfere with the 15 host cell's . ~ and cam be ,' ' ' using any of a variety of cell-free 'i ' systems that are known in the art. GDFPs can also be prepared a,~ . It will likely be desirable for the GDFP to possess a component or sequence that can facilitate the detection and/or L r- '' of the GDFP. Such a component may be the same as or different from one of the various 20 described above.
Many approaches of expressing and purifying ' proteins are known to those skilkd in the art, and kits for r ' protein expression and l ~
are available from gveral r ' of molecular biology products.
Typically, an increased level of purity of the GDFP will be desirable. HoweYer, 25 because of the specificity of the GDFP for nuckic acid binding, the degree ofneed not necessarily be extensive. The GDFPs of the present invention may be sterili7ed by simple filtration through a 0.22 or 0.45u filter so as to avoid microbial, of the target cells.
Since the domains of the GDFP can be assembled in modular fashion in an 30 expression vector, its ,.)~ ;..., by lC ' DNA . ~ ;.r allows the GDD
to consist of one or many ~ Such: ~ may have , ' activity in mediating or enhancing gene delivery, or they may have closely related WO 9~/28494 2 ~ 8 7~ l 8 PCTIUS9~/04738 functions. In essence, the gene deliYery domain can be viewed as possessing any fimction that mediates or enhances the efficiency of delivery of the ~NA bound to the GDFP.
S Other Variations of GDFPs ~ .
Other variations will be apparent to those of skill in the art. For example, theGDFP may itself be, ll;" ;,. .l M~ -- may be a.l~ _ to increase avidity of binding of either the NBD or the GDD. A given tNA molecule may also contain multiple distinct cognate recognition sequences, binding different Type l 10 GDFPs with distinct functions, or the tNA may be bound with a mixture of Type I and Type 11 GDFPs. Additionally, certain ~ " of tbe GDD, such as IN proteins, may require ,l " . ;~;.... for optimal activity. D of the GDFP may be obtained by including, for example, a leucine zipper motif in the GDFP. Such motifs are common in DNA binding proteins and are responsible for their ' (Trr,~l7~ritlPc 8~ Ziff, 1989). Leucine zippers can be inserted into DNA binding proteins and cause them to dimerize (Sellers and Struhl, Nature 341:74-76, 1989).
l\' ' of GDFPs can also be achieved, for example, by creatirlg a IC ' ' ' fusion protein tbat contaios two or more GDFPs. Preferably such ' ' GDFPs are separated by flexons, as described herein. Other 20 ol;~;- ; -;;~ motifs from dimeric or multimeric proteins can similarly be employed.
Illu ~ratiorc of T~n Gene l)eliven Ebsion Protei~2 Type-ll GDFPs do not bind targeted nucleic acids in a sequence-specific maoner 2S because the nucleic acid binding ~ of Type-ll GDFPs are all derived from nucleic acid binding proteios that are ~on-sequence-specific in their binding to nucleic acid.
~llrlrir ,Arirl Rin-iin~ Tn~ilK of Type-~ GDFPs The nucleic acid binding domairls (NBDs) of Type-ll GDFPs comprise binding that are derived from non-sequence-specific nucleic acid binding proteins, fused to a gene delivery domain (GDD) as described above.
.

. _ . . . . .. . . .. .. . .

WO 95128494 2 1 8 7 ~ 1 ~ r~v~ -~ 1738 A number of non-sequence-specific nucleic acid binding proteins have been identified and ~ , r~ 1, including, for example, histones or pC~ D deriYed therefrom (see, e.g., von Holt, Bioassays 3:120-124, 1986; Rhodes, Nucleic Acid Res., 6:1805-1816, 1979; and Rodriguez et al., Biophys. Chem., 39:145-152, 1991~;
retroviral, l~J ~ l proteins (see, e.g., Gelfand et al. J. Biol. Chem., 268:18450-18456, 1993); proteins such as nucleolin (Erard et al., Eur. J. Biochem. 191:19-26, 1990); avidin (Pardridge & Boado, F.E.B.S. Lett. 288:30-32, 1991); and polybasic~ul.~ Li~ sequences such as poly-L-lysine (Li et al., r- ~ y, 12:1763-1772 1973; Weiskopf and Li, Biu~ul~ D 16:669-684, 1977).
For the reasons discussed herein, all of the GDFPs of the present inYention are preferably produced as fusion proteins. However, the l~
expression, in a host cell, of non-sequence-specific nucleic acid binding c~ in Type-lI GDFPs (as well as in Type-I GDFPs tbat incorporate , non-specific nucleic acid binding , ) may be hindered by r of the expressed proteins with host cell nucleic acids. In such situations, the GDFPs can be readily synthesized in vitro using any of a variety of cell-free ~ systetns that are known in the art.
r~n~ Deliverv Domains of TvDe-ll GDFPs The various possible sources of . , making up the gene delivery domains of Type-lI GDFPs are essentially the same as described above for Type-I
GDFPs (although, by deftnition, Type-II GDFPs would not include sequence-specific binding c~ q-~ such as the sequence-specific integrase , described above for Type-l GDFPs).
Tareeted ~ r Acids for Use with Tvpe-ll GDFPs The t~rgeted nucleic acids to be combined with Type-lI GDFPs are as described above except that they need not contain specific recognition sequences since the Type-ll GDFPs bind nucleic acids via non-specific W095128494 2~g7~?8 r~ ol738 ~

,A~cPmh~y of TvF~e-lI GDFPs The assembly of Type-ll GDFPs is preferably via the synthesis of ~c.ullll, fusion proteins (see the description above regarding assembly of Type-l GDFPs).
S ~l~inv GDFPs of/he Present InvPntiF~n Thus, the GDFPs ûf the present invention can be used for in vitro or in vivo gene delivery. For tberapeutic -~ target cells can be transduced ex vivo and returned to a patient, or, given the 1;~ nature of tbe tNA/GDFP complex, cells can be treated directly in vivo. For such in vivo therapy, the complexes can be for~nulated for a variety of modes of - including systemic and topical or localized ' Techniques and r " may be found, for example, in R ~s PI~ ciences. Mack Publishing Co., Easton, PA. (latest edition). The tNA/GDFP complex may be combined with a carrier such as a diluent or excipient which may mclude, for example, fillers, extenders, wetting agents, ~' ~ , sutface-active agents, or lubricants, depending on the nature of the modeof ' and the dosage forms. The nature of thc mode of F ' ' ' ' wi depend, for exatnple, on the location of the desired target cells. For in vivo injection is preferrd, including ', 1, illLla~Lluu ~, intra-arterial (including delivery by use of double balloon catheters), ;.I;~ and ' Delivery to lung tissue can be ~ I by, e.g., ~1~ ' For injection, the complexes of the invention are formulated in liquid solutions, preferably in 1', ", "~, compatible buffers such as Hank's solution or Ringer's solution. In addition, the complexes may be formulated in solid form and redissolved or suspended ' '~ prior to use. Lyophilized forms are also included. Systemic - ' can also be by i ' ûr means, or the compounds can be ' ' orally. For i ' or 1.~ .~ l ", r ' ' ' ' penetrants appropriate to the barrier to be permeated are used in the r""""1 l ;. .1~ For topical ~ ' the complexes of the invention may be formulated imto ointments, salves, gels or creams, as is generally known in the art.
The GDFP approach cam thus be used to target any cell, in vitro, ex vivo or in vivo, the only ICU,UiI~ being that the target cells have binding sites for the GDFP
on their surface. The present invention will thus be useful for many gene therapy 2~878t~
wo gs/28494 1 r~ c t73x ~5-As an illustralive example, the target cells that could be used in the context of the present invention include Iyl..~ cells. These include: (i) slem c-lls, which have many potential A~ ;..-- in gene therapy, including corection of hereditary disorders such as Gaucher disease and 1- ~ ~lyl-ll-;----~, I. ~ as 5 well as genetic ~, r~ . with inrr-r~ r vaccines against HIV such as decoys or ' negative proteins; and (ii) Iy . ' ~ O, which would allow genetic "...,1;1-;, ~1;ll.. of effector T cells such as CTLs for use in human theraw with genes of inurest such as suicide genes and regulated promoter cassettes. Also included for use in the context of the present invention are cells of the ~diu~a~.ul~ system which line 10 blood vessels including endothelial cells and vascular smooth muscle cells, which could be genetically modified to inhibit dtl~ ua~ ua;a or restesis fûllowing dll~;;uyL aiy.
Similarly, the present invention could be used to introduce genes into airway epithelial cells, such as the CFTR gene to ccrect cystic fibrosis. The present invention could also be used tû transduce tumor cells and thereby genetically modify them to express 15 suicide genes for tumor elimination or produce cytokines or express y molecules for use as a tumor vaccme in cancer patients. Another illustrative application of the present invention is delivery of DNA ûr RNA to antigen presenting cells (APCs). This could be useful, for example, to allow expression of specific (tNA-encoded) antigens by an APC, thereby allowing the APC to stimulate an antigen-20 specific immune response, such as a CTL response. Such an approach can be used invitro, by i of APCs with a GDFP/tNA complex thereby allowing antigen ;,... for the stimulation and generation of CTLs in vitro, or in vivo delivery can be used, to allow such antigen ~ in vivo. Direct delivery ûf RNA to APCs using the present invention may be especially desirable for situationâ in which 25 antigens are encoded by transcripts that require special conditions for " 'transport or processing that may not happen efficiently in the APC. An illustrative example would be rev-dependent RNAs of HIV (such as HIV gag). T of APCs with RNA in the context of the present invention can thus be used, for example, to circumvent the need for nuclear export of rev-dependent RNAs. Additionally, the 30 present invention could be used to introduce genes into ~, ~.-.. of the liver to correct genetic defects such as familial ll.y~ .1 ul~...;~, hemophilia and othermetabolic disorders, or to produce ,~...,.I, --.I products for systemic delivery.

2 1 878 1 ~
WO9~i/28494 1"~~ 5/.1738 Similariy, fibroblasts or conneclive tissue cells could be modified to secrete cytohnes or soluble enzymes for ;, . ~ purposes or to cotrect a metabolic deficiency. These tissue targets and diseases, together with others are more fully described in Scriver et al., Eds., 'Tne Metabolic Basis of Inherited Disease', 6th Ed., S McGraw-Hill, 1989, and in Miller, A.D., Blood 76:271-278, lg90. Tile present invention is I ' '~, useful in cases in which genes of interest cannot be transferred by commonly used viral vectors, or in which the target cells are not infectable by viral approaches (see, e.g., Israel & Kaufman, Blocd, 75:1074-1080, 1990; ~' : ' , &
Temin, Nature 299,265-268, 1982; Stead et al., Blood, 71:742-747, 1988; and Bodine et al., Blood, 82:1975-1980, 1993).
The GDFP approach of the present invention can be used as a generically useful method for geDe of cells, and could be provided as a laboratory kit for gene 1 "-~ for use with, e.g., insect, avian, " or other higher eukaryotic ceils.
The transfer of genes m the present invention can also be facilitated by other t ~l~ known to enhance the uptaice of rlucleic acid by cells (see, e.g., Kawai &Nishizawa, Mol. Cell. Biol. 4:1172-1174,1984; Behretai., P.N.A.S. 86:6982-6986, 1989; Rose et al., P.N.A.S. r 10:52~525, 1991; Pardridge & Boado, F.E.B.S. Lett. 288:30-32, 1991; Legendre & Swica, P.N.A.S. 90:893-897, 1993;
Haensler & Swi~a, Bioconj. Chem. 4:372-379, 1993). These and other techniques for use in the conte~t of the present inverltion can be used under conditions (for incubation etc.) as described in tne art (see, e.g., Kriegler, M. 1990 (ed.), "Gene Transfer and ~pression, a Laboratory Marmal," (1990)). In the case of GDFPs comprismg pH-dependent M-D r ' . such as the TM proteim of diphtheria toxin (see, e.g., Choe et al. (1992) Nature 357:216-2æ), entry of the GDFPltNA complex mto the cell can be ,,u..~ achieved by simply reducing the pH of tile incubation medium during ( ., l. .. l ;~ .. .
The examples presented below are provided as a further guide to the ~
of ordinary skill in the art, and are not to be construed as limiting the invention m any 30 way.

WO 95128494 2 1 8 7 8 ~ 8 P_ I ~u~ ~738 -4~-Exam~le I
Preparation of a Nucleic Acid Rinriin~ Domain (NBD) From the Yeast G~r 4 r 'rhe DNA binding domain of GAL4, amino acids 1-147 (Laughon and Gesteland, Molecular and Cellular Biology 4:260-267, 1984; Ma and Ptashne, Cell 48:847-853, 1987; and Carey et al., J. Mol. Biol. 209:423-432, 1989), was amplified by PCR from S. cerevisiae (ATcc 60248) using the following amplimers.
The amplimer for the 5' end of GAL4 was as follows:
5' GCGC ACTAGT GCCACC ATG AAG CTA CTG TCT TCT ATC G 3'.
The GAL4 coding region is umderlined. This amplimer created a Spel site (ACTAGT) for cloning mto r~- . (stratagene) which allowed for subsequent by T3 RNA pul~ The amplimer also included a consensus sequence (GCCACC) for efficienî protein translation located upstrearn of tbe initiator methionine (Kozak et al., Nucl. Acids Res. 15:3374, 1987).
Tbe amplimer for the 3' end of tbe GAL4 NH2-terminus (up to amino acid 147) was as follows:
5' GCGC GGTACC TCCGGA TAC AGT CAA CTG TCT TTG ACC 3'.
Tbe GAL4 coding region is underlined. This amplimer created a 3' Asp718 site (GGTACC) for cloning into i '`' , as noted above. The amplimer also included a BspE1 site (TCCGGA) to allow for an in-frame fusion witb an oligomer encoding a flexible peptide sequence (see below).
The GAL4 fragment was amplified by 30 cycles of PCR directly from a colony of S. cerevisiae. Tbe product was digested witb Spel and Asp718 and ligated between the Spel and Asp718 sites located in the polylir~cer region of E'" , The construct was ~ r "~ -- 1 into the DHlOB stram of E. coli by ~ L u~u~Liul~, and a colony containing tbe GAL4 fragment was identified by restriction enzyme analysis.
The resulting plasmid, designated pT3gGAL4, is shown in Fig. 2A.

21 8781 ~
WO 9S128494 r~,l/u~ !O ~738 l~am,~le 2 Pre7~aration of a Genç Deliverv D-~mAin From ti e ~117~7An lL-2 Pr~i77 A DNA fragment encoding mat77re spluble human IL-2 ~amino acids 21-133) 5 was amplified by PCR from a full-length human IL-2 cDNA (Taniguchi et al., Nature 302:305-310, 1983), using the following amplimers.
The amplimer for the 5' end of mature huma~ lL-2 was as follows:
5' GCGC ACTAGT GCCACC ATG GCG CCT ACT TCA AGT TCT ACA AAG
~3'.
The IL-2 coding region is underlined. This amplimer created a Spel site (ACTAGT) for cloning into rRI7~5r7 irt and inserted an initiator methioni~e - ' '!! upstream of atnmo acid 21 of IL,2. The amplimer a so contained a consensus æquence ~&CCACC) for efficient translation upstrea7n of the i~serted , as Loted above. A Narl site (GGC&CC) was a77sP included which allowed for a subsequent in-fra7ne fusion with a inker sequence which separated the GAL4 and IL-2 domaiLs in the GAL4/lL-2 construct {see below).
The amplimer for the 3' e~d of mature human IL-2 was as follows:
5' GCGC GGTACC TCA AGT CAG AGT ACT GAT GAT &CT TT& ACA AAA
&GT AAT C 3'.
This amplimer created an Asp718 site (GGTACC) for clonimg into rR~ sr~irt, And a so retained the wi d-type i codon for human IL-2. A Scal site (AGTACT) at the 3' end of the IL 2 coding region was also creat7-d by this amplimer without introducing amino acid cbanges. The DNA fragment encoding the mature human IL-2 prPtein was amplified by 30 cycles of PCR from the full-length human IL-2 cDNA referred tP above. The prPduct was digested with Spel and Asp718, ligated into rRl~ sr~ir7. and ~ f ~ i into DHlOB cells as described above. A
colorly harbormg an appropriate constAuct was identified by restriction enzyme analysis.
Sequencing of a plasmid derived from one colony revea77ed that an a7teration (loss of a single base rerulting in a frame-shift near the terminus of IL-2) had occurred within the 3' amplimer during PCR clonin~ - thereby generating an IL-2 mutein.
Specifically, the frst T after the Sca I site (in 7he ftrst GAT triplet) was removed, causing a frame-shift that a77so generated a premature i codon. As a result, _ _ _: _ _ : . , . . . . ... . _ _ . ... . . . . . . . .

2187~i~
WO 95128494 r~ 0.1738 the 5 amino acids normally present at tne terminus ~vere replaced by 3 different amino acids. This plasmid, referred to as "pT3matrL-2m" (shown generically in Figure 2A as pT3matrL-2), was used to create a gerle delivery fusion protein as described in Example 3. Despite the variation in the IL-2 domain, a GDFP based on this IL-2 5 mutein exhibited IL-2 bioactiviy, as described below.
A second colony contained a plasmid desigrlated ~pT3matlL-2" (as shown generically in Figure 2A) that contained the expected wild-type rL-2 sequence.
Plasmid pT3matrL,2 was used to create two GDFPs as described in Exarnples 3 and 4.
.Ex~le 3 Co~ of p~ c Fn-~in~ a Gene Deliverv Fusion Protein (GDFP) Havi~ a GDD and an NBD
~Ç~a~Led bv a F~ rr.n A DNA fragment encoding the nucleic acid binding domain (NBD) derived from GAL4 was isolated from pT3gGAL4 (Example 1) by digesting with Spel and BspE1.
A DNA fragment encoding the gene delivery domain (GDD) deriv~d from a human rL~2 mutein was isolated from pT3r~atlL-2m (Example 2) by digesting with Narl and Asp718. The following oligomer pair encoding tne flexon sequence (GlyGlyGlyGlySer)3 was annealed creating a 5' BspEI over-hang (CCGGA) and a 3' 20 Narl over-hang (CGCC):
5' CCGGA GGC GGT GGA TCC GGT GGT GGA GGC AGT GGA GGA GGT GGC
TCGG 3';
5' CGC CGA GCC ACC TCC TCC ACT GCC TCC ACC ACC GGA TCC ACC
GCC T 3'.
The NBD and GDD fragments and the annealed oligomer were ligated into pBluescript between the Spel and Asp718 sites, and i ~ ' into DHlOB cells as described above. A colony harboring a construct that contained all three fragments was idertifled by its ability to hybridi7e to both GAL4 and IL-2 [32P]-labeled fragments, and by restriction en7,yme analysis.
In the resulting plasmid, designated "pT3GAL4/lL-2m" (shown generically in Fig. 2A as pT3GAL4/lL-2), the sequence erlcoding the GDFP was inserted irlto pBluescript in an orientation which allowed for sense RNA transcripts to be synthesi7ed 21878t~
WO gS/28494 P~ S10~738 with T3 RNA polymerase. The resulting RNA, when trans~ated, iII.,UI; ' ' bûth the DNA binding domain of the yeast GAL4 protein and the mature form of the human IL-2 mutein, in that order, separated by a flexible amino acid lirlker.
A second plasmid, designated pT3GAL41IL-2 (as shown in Fig.2A), was 5 corlstructed exactly as described for pT3GAL41IL-2m, except that the DNA fragrnent encoding the gene delivery domain (GDD) derived from human IL-2 was isolated from pT3matlL-2 (Example 2).
Example 4 Conc~l~ti~n of a Third Pl~ Fnrntlin,~ a Gene Delivery F~ n Protein (GDFP) HavinF a GDD and an NBD
~ted bv a Flexon Another expression vector encoding a GDFP derived from IL-2 and GAL4 was constructed as follows.
The DNA binding domain of GAL4, amino acids 1-147 (Carey, et al., supra), was amplifled by 30 cycles of PCR from pT3gGAL4 using the following amplimers.
The amplimer for the 5' end of GAL4 was as follows:
5' GCGC GGATCC ATG AAG CTA CTG TCT TCT ATC G 3'.
This amplimer created a BamHl site (GGATCC) ~ 1~, upstream of Met~
to allow for an in-frame fusion witb a flexible peptide sequence in frorlt of GAL4 (see below).
The amplimer for the 3' end of the GAL4 NH2-terminus (up to amino acid 147) was as follows:
5' GCGC GGTACC G CTA GCT TAC AGT CAA CTG TCT TTG ACC 3'.
This amplimer created an Asp718 site (GGTACC) for cloning into pBluescript and also included an engineered i codon (CTA) at the C-terminus of the DNA binding domain of GAL4.
To construct pT3IL-2/GAL4, the GAL4 PCR product was digested with BamHl and Asp718. A DNA fragment encoding human IL 2 was isolated from pT3matlL-2 (see Example 2) by digesting with Spel and Scal. The following oligomer pair encoding the ammo acid sequence (GlyGlyGlyGlySer)3 was annealed, creating a 5' Scal over-hang (ACT) and a 3' BamH1 over-hang (GATCC):
_ _ . . . _ .. . ... . . . . . ..

t ~
WO 95/28494 -51- P~ J~738 5' ACT CTG ACT GGA GGT GGG GGC TCT GGT GGC GGA GGT AGT GGA
GGA GGT G 3';
5' GA TCC ACC TCC TCC ACT ACC TCC GCC ACC AGA GCC CCC ACC TCC
AGT CAG AGT 3'.
The IL-2 and GAL4 fragments and oligomers were ligated into pBluescript between the Spel and Asp718 sites and the construct was L~ ru~ ~i into the DHIOBstrain of E. coli by el.~LIu~ iull. A colony containing all three fragments was identified by its ability to hybridize to both GAL4 and IL-2 [3 P]-labeled fragments, and by restriction enzyme arlalysis.
In the pT3I~2/GAL4 construct, shown in Figure 2B, the GDFP was inserted into pBluescript in an orientation which allowed for a sense RNA to be synthesized with T3 RNA p~l~ The resulting RNA, when translated, ill~OI~ ' botb the mature form of human IL-2, and the DNA binding domain of the yeast GAL4 protein,in that order, separated by a flexible amino acid linker.
Example 5 FYprE ~ )n of Gene Deliven Fusion Prnh~inr~
Sense mRNA encoding the GAL4/lL-2m GDFP corlstruct (described in Example 3) was transcribed in vitro with T3 RNA polymerase from the pT3GAL4/lL-2 vector.Briefly, pT3GAL4/lL,2m plasmid was linearized witb Asp718 and this template was combined with a l-l ' ' mixture (rNTPs), RNA cap structure analog (m7Gppp), and T3 RNA p~ in a HEPES-based buffer ¢romega "RiboMAX~).
After incubation at 37 degrees C, the DNA template was digested with RNase-free DNase (Promega), and the synthesized mRNA was separated from l rNTPs by ' ' ,, ,' ., through a G25 Sephadex spin column (I' ' _ Mannheim), ~ . ' with EtOH, and quantitated by OD260.
The resultant mRNA was trarlslated in a cell-free rabbit ' ~.. Iysate system. mRNA was added to a translation mixture of l~t;.ul~ .. Iysate, RNasin, and complete amino acids (Promega). Translation was allowed to proceed for I to 2 hr. at 30 30 degrees C, after which Iysates were stored at -70 degrees C. The integrity and molecular weight of the fusion protein was assessed by including [3~S]-labeled methionine (Amersham) in the translation mix, and visualizing the product by W0 95/28494 2 1 8 7 8 1 8 P~IIIJL ~ I738 .

pol~ ..ide gel el~ upllul~DiD under denaturing conditions. Fig. 3, lane 2, showsthe [35S]-labeled GAL4/~L-2m translation product as resolved on a 14% acrylamide gel.
The position of the GAL4/Ik2m GDFP translation product agreed with the predictedMW of 33kD. Molecular weight markers ate shown in Fig. 3, lane 1, and a negative5 control is shown in lane 3.
Sense mRNAs encoding the GAL4/~L-2 &DFP construct (described in Example 3) and the IL-21GAL4 GDFP construct (described in Example 4) were transcribed invitro with T3 RNA polymerase from the pT3GAlA/IL-2 and pT3IL,2/GAL4 vectors, ICD~ ,IJ, exactly as described above. Figure 6, lanes 3 and 4, show the [35S]-labeled IL-2/GAL4 and GAL4/IL-2 translation products as resolved on a 4-20%
gradient acrylamide gel. The positions of the IL2/GAL4 and GAL4/lL-2 GDFP
t~anslation products agreed with the predicted MWs of 33.3kD aod 33.2kD, I~D~ Molecular weight markers are shown in Fig. 6, lane 1, and a luciferase control is shown in lane 2.
Exam~le 6 Se~tuence-Specific DNA Rinrtinri Activity of GDFPs The ability of the GDFPs of Example S to engage m . ~i~l~, DNA
binding was l' ' by use of an ~ t ~ ;;. mobility shift assay (EMSA) (Ausubel et al. (eds), ~Current Protocols bn Molecular Biology,~ (1987 and 1993)).
The target oligomer to which the GAI,4 protein binds was:
5' TCGACGGAGTA~ CGC 3' 3' GCCTCATGACAGGAGGCGAGCT 5'.
The following target oligomer is not bound by GAL4 and was used as a negative control:
5' TCGACTGAGTA~ AGC 3' 3' GACTCATGACAGGAGTCGAGCT 5'.
The GAL4 target oligomer was end-labeled using [3ZP]-dCTP (Amersham) and Klenow polymerase tNew Englamd BioLabs). The labeled oligomer was separated from I . ' nucleotides by ~.hl, O . ' ~ over a G25 spin column h in~ r~ ~ ) and quantified by " cour~ting. This oligomer was added to reactions containing a HEPES-based buffer, which mcltlded ... , .: _ .. ... ......... ... ... _ .: . _ _ . .. _ _ .. _ _ = _ _ _ _ = _ _ _ .

WO 95/28494 2 ~ 8 7 ~ 1 ~ PCI/lJS9~;/04738 poly(dI-dC)-poly(dI-dC) (Pharmacia), ZnCI~, glycerol and BSA (Carey, et al., J. Mol.
Biol. 209:423432, 1989; Chasman, et al., Mol. and Cell. Biol. 9:47464749, 1989),and varying amounts of reticulocyte Iysate containing either GAL4/IL-2m GDFP or IL-2. The reactions were Cl.,~L~u~llulc~cd on a 4.5% p~ l;d~/1% glycerol gel in O.Sx TBE. The gel was fixed in a methanol/acetic acid solution, dried, and analyzed on a Molecular Dynamics 1' , ' ~ . The results shown m Figure 4 show decreasing amounts of input GAL4/IL-2m fusion protein (lanes 1-3) showing specific interaction of the GAL4/IL-2m GDFP with the labeled target oligomer. DNA size markers appear in lane 4. In lane S, the GAL4/lL-2m GDFP was incubated with labeled target oligomer, as in lane 1, but excess unlabeled target oligomer was also included and competed with the labeled target oligomer for binding to the GAL4/lL 2m GDFP. In lane 6, the GAL4/lL-2m GDFP was incubated with labeled target oligomer,as im lane 1, but excess unlabeled non-bindimg oligomer was included, and showed lack of I , for binding of the GAL4/lL-2m GDFP to the labeled target oligomer.
In lane 7, labeled target oligomer was incubated with Iysate containing human IL-2, and showed no specific bindmg of the labeled target oligomer by either IL-2 or le~i.ulu~rt. Iysate . . The GAL4/lL 2m GDFP thus boumd, ~ "~, to the cognate target sequence recognized by the GAL4 (NBD) domain of the GAL4/IL-2m GDFP.
The results im Figure 7 show sequence-specific binding of the GAL4 protein and the IL-2/GAL4 and GAL4/lL-2 GDFPs. Target oligomers, bmding conditions, cl.. ,' and gel fflatment were exactly as described above, except that analysis was by ' ~. The frst four lanes contained decreasing amounts of mput GAL4 protein, as indicated, showing specific interaction of GAL4 with the labeled 25 target oligomer. The following lanes contamed decreasmg amounts of either input GAL4/IL-2 GDFP or input IL-2/GAL4 GDFP as indicated in Figure 7. The ~l~ci~n~ n " +Cn indicates that the GDFP was incubated with labeled target oligomer, as im previous lanes, but excess unlabeled target oligomer was also mcluded in the reaction. The designation " +m" indicates that the GDFP was incubated with labeled 30 target oligomer, as in previous lanes, but excess unlabeled nu.. ~ ~ oligomer was included in the reaction. The unlabeled target oligomer competed with the labeled target oligomer for bmding to the GDFP while the non-binding oligomer showed lack WO95/28494 21 87 81 ~ r~l~uv 01738 .

of ~ ", ~ r, specific binding of the GDFP to the GAL4 recognition sequence. In the lane designated "IL2,~ labeled target oligomer was incubated with Iysate containing human IL-2, and showed no specific binding of the labeled target oligomer by either IL-2 or reticulocyte Iysate ~.. l.. -t~ The IL-2/GAL4 and GAL4/lL-2 GDFPs thus bound specifically to the cognate target sequence recognized by the GAL4 ~NBD) domain of the GDFPs.
Example 7 CVtr~kin~ Bioactivitv of GDFPs GAL411L-2m, GAL4/lL-2, and IL~2/GAL4 fusion proteins from in vitro (as in Example 5) were assayed for their IL-2 activities using the well-known CTLL bioassay. Cells were incubated with the GDFP, then pulsed with 3H-thymidine and i.. ~ of l_di~ ivily into DNA was used as a measure of cellular pl~ ~-r '- , as described by Gillis et al., J. Immunol. 120:2027, 1978.The results from the GAL4/lL-2m GDFP are shown in Figure 5. The IL,2 standard represents I ng/ml human IL-2 which was serially diluted 1:3 in the bioassay. The GAL4/IL-2m GDFP curve was generated using in vitro translated material starting with a 1:10 dilution of Iysate. The bioassay shows retention of IL-2 biological activity by the GAL4/IL-2m GDFP.
The results from the GAIA/IL-2 and IL,2/GAL4 GDFPs are shown in Figure 8.
The IL-2 standard was as described above. The GDFP curves were generated using in vitro translated material starting with a 1:50 dilution of Iysate. The bioassay shows reterltion of IL-2 biological activity by the GAL4/ILr2 and ILr2/GAL4 GDFPs.
E~ample 8 (~ImCtnlrtifm ~ (~ ''AI .. .;~AI;l~.. of GDFPs ~on~ i~ the Dinhth~i:~ Toxin ~ Anr R~vi~n The i ' (TM) domain of the Diphtheria toxin protein from C.
rlirhth~ amino acids 205-378 (Choe, et al., Nature 357:216-222, 1992), is the 30 region resporlsible for endosomal release of the catalytic domain of the toxin into the cytoplasm of infected cells (Papini, et al., JBC 268:1567-1574, 1993, Madshus, JBC
269:17723-17729, 1994). This region has also been shown to be capable of cellular Woss/28494 . 2 ~ 8 78 ~ 8 r~.~u~ ~ ~4,~8 .

membrane insertion in response to a mildly acidic ~llYilU.~ t (Moskaug, et al. JBC
263:2518-2525, 1988, McGill, et al., EMBO 8:2843-2848, 1989). To incorporate this domain into the pre-existing GDFPs, two DNA fragments encoding the ~
domain of the Diphtheria toxin protein were amplified by PCR from C. dit~htheriae genomic DNA. The first fragment, termed "DT," encoded amino acids 205-378 and was amplified using the following amplimers.
The amplimer for the S' end was as follows:
5' GTAGATCTGGTGGAGGTGGCTCCGGAGGAGGT GGATCC GAT TGG GAT
GTC ~ AGG 5~ _ 3' The amplimer for the 3' end was as follows:
5' CTTC AGATCT GGATCC T CCA CCG CCA CTA CCT CCA CCC CCG GGA
CGA TTA TAC GAA TTA TGA ~ 3' The toxm TM sequences are underlmed. ~oth amplimers provided BamHI sites near the termini for subsequent cloning of the PCR fragment into BamHI-digested pT3IL-2/GAL4 (Example 4).
The second fragment, termed "DAB," encoded amino acids 176-378, and provided additional residues at the amino terminus of the i ' region.
Within the cont~%t of the mtact toxin protein these additional sequences are mvolved in an enzymatic cleavage step which may be necessary for membrane fusogenic activiq(Williams, et al., JBC 265:20673-20677, 1990, Ariansen, et al., r ~ y 32:83-90, 1993). The ~DAB" fragment was amplified using the following amplimers:The amplimer for the 5' end was as follows:
5' GCG GGATCC GGT GGC GGA GGA AGT GAT GCG ATG TAT GAG TAT
ATG ~ C 3' The amplimer for the 3' end was the same 3' amplimer used to PCR the above described "DT" fragment.
The toxin TM sequences are underlined. The resulting "DAB" PCR fragment was digested with BamHI, amd cloned into the BamHI sites of both pT3GAL4/IL-2 and pT3IL-2/GAL4 (Examples 3 amd 4).
Thus, three triple-domain fusion plasmids were generated (pT3IL-2DTGAL4, pT3IL-2DABGAL4, and pT3GAL4DABIL-2), each containing a version of the diphtheria toxin ~ region as the middle domain. Each of the three GDFP

2 ~ 8? B ~ 8 WO9~/28494 -56- lE~"~ ~o 1738 RNAs was translated and the resulting fusion proteins were assayed for reteMion of IL-2 bioactivity and specific nucleic acid binding abiiity (Examples 5, 6, and 7). Ail three triple-domain GDFPs were found to retain these activities.
Exami?le '~
Construction of a T:~reeted Nucleic Acid (tNA) The yeast ~ t;~ ' activator, &Ai'A, has specific affirtity for severai closely related 17bp double-sttanded DNA sequences, and it has aiso been shown to bind consensus synthetic 17bp target sequences with a similar afftnity as it does wild-type sequences (Giniger, et al., Cell 40:767-774, 1985; Bram and Kornberg, P.N.A.S. 82:4347, 1985). Target vectors were made by ligating oligomers containing a consensus 17bp sequence (Webster, et al., Cell 52:169-178, 1988; Carey, et ai., J.
Mol. Biol. 209:423432, 1989) into the unique Sail site in the backbone of pDC302CAT (Mosley et al., Cell 59:335-34~, 1989; and Overell et ai., J. Imm. Meth.
141:53-62, 1991), a plasmid which directs the expressiorl of ;' ' .' ' acetyl transferase (CAT) m " host cells. The following oligomers were used:
5' TCGACGGAGTACTGTCCTCCGC 3' 3' GCCTCATGACAGGAG&CGAGCT 5'.
The oligomer pair harbored an iMernai Scal site (TCATGA) witich was used to 20 screen resuiting 1, ---- r-",---t~ for presence of the oiigor~ter. in addition, there were Sall-compatible over-hangs at both the 5' and 3' ends, oniy one of which could regenerate the Sall site upon ligation. This feature was ~ull ' to allow the release of oligomer muitimers for plasn~id ,~ - The oligomer pair was anneaied by boiiing equai amounts of each in a modeate sait buffer, then slow cooling the reaction. The anrleaied oligomers were then icinased with T4 prJI~,,~I~u~ile kirtase (Ro~hrtn~r Mannheim) and ligated to pDC302CAT which had been linearized with Sail. The consttucts were ~ u~. - ' into DHlOB ~ coli amd resultmg colonies were screened for the presence of the target oligomer, or multimers thereof, by 11, ~li ii~liu.. to a [3ZP]-labeled target oligomer probe. Color~ies harboring the target 30 oligomer were further ~ I for number of copies by restriction enzyme arlalysis and ~r.~ nrin~ ~

W0 95128491 2 1 8 7 8 1 8 r~ 73s .

F~y~mnlP 10 Abilitv of GDFPs to Bind to JT.-2 ReceDtor-R~o~in~ CTLL
GAL411L-2 and IL-2/GAL4 GDFPs from in vitro translations (Example 5) were further ~I~ml ' tO bind CTLL (see Example 7) via the following assay. CTLL
were incubated in IL-2-free medium for 2 hours or ionger. [35S]-labeled GAL411L-2, IL-21GAL4, IL-2, and GAL4 (Example 5) were incubated with the CTLL for 1 hour al4 degrees C in a binding medium containing 25mglmi BSA and 2mg/mi Na-a7,ide in RPMI-1640 buffered with 20mM HEPES. The binding medium was adjusted to a final pH of 7.2 prior to use. After binding, the cells were washed tilree times in ice cold PBS, and the final cell pellet was 1~ . ' ' in a Tris buffer containing 150mM
NaCI, 5mM EDTA, 0.02% Na-azide, and 0.5% Triton X-100 to gentiy Iyse the cells.
The Iysate was spun briefly, and the ~ was Cl.~ll, .' ' tbrough a 4-20%
gradient ~vl.~a~ly' ' gel. Figure 9 shows labeled protein present in the CTLL
Iysate and, therefore, associated with the CTLL. Lane I shows a molecular weightstandard. Lane 2 shows the human IL-2 protein as present in the unreacted ~ uiu~Iysate (Example 5), and lane 3 is the CTLL Iysate after binding to IL-2. Lane 4 shows the GAL4/lL-2 GDFP as present in the unreacted lefi.,.~ t. Iysate, and lane 5 is the CTLL Iysate after binding to the GAL4/lL-2 GDFP. Lane 6 shows the II~2/GAL4 GDFP as present in the unreacted .~ ulu.~ lysate, and lane 7 is the CTLL Iysate after binding to the ~L-2/GAL4 GDFP. Lane 8 shows GAL4 as present in the unreacted l~ .Ui~.~ Iysate, and lane 9 is the CTLL Iysate after binding to GAL4.The GAL4/IL-2 and IL-2/GAL4 GDFPs and IL-2 thus bind specificaily to CTLL while GAL4 does not. This !' that CTLL-specific binding of the GDFPs is mediated by the IL-2 domain and not by the GAL4 domain.
ExamDle 11 Abiiirv of GAL411L-2 and JT~2lGAL4 GDFPs to Media~jS~j~ of a Tar~l t Oli~omer to n.-2 ReceDtor-BearinF CTT.T.
GAL41IL-2 and IL-21GAL4 fusion proteins from in vitro translations (as in Example 5) were bound to [32P]-dCTP-end-labeled GAL4 target oligomer as described in Example 6. The GDFP-tNA complex was bound to CTLL, as described in Example 10, for 1 hour at 4 degrees C in binding medium containing 25mg/mi BSA and 2mglmi W0 95/28494 2 1 8 7 8 1 ~ P~ O I738 .

Na-azide in RPMI-1640 buffered with 20mM HEPES. The binding medium wasadjusted to a final pH of 7.2 prior to use. The cell-bound GDFP-tNA complex was separated from free GDFP-tNA by ,~ ;r"~ ., of tne binding mixture through a phthalate oil layer (Dower, et al., J. Exp. Med. 162:501-515, 1985) Cell-associated counts were quantified by sri~-illqtir~n countulg. Figure 10 shows counts of labeled oligomer associated with CTLL as mediated by the GAL4/lL-2 GDFP, the IL-2/GAL4 GDFP, GAL4, and a negative control l~li.ul~i~ Iysate designated "Bg." The binding assay ~' tne ability of both GAL41lL-2 and IL-2/GAL4 GDFPs to mediate binding of the oligomer tNA to CTLL.
F ' 12 ~kility of the GAL41rT~2 GDFP to Mediate Bindine Of a Tar~et Plasmid to IL-2 Receptor-Beariruc~aLL
The GAL4/lL-2 GDFP (Example 5) was bound to the target plasmid using binding conditions ciescribed in Example 6. The plasmid contained eight copies of tne &AL4 17-mer target oligomer, as described in Example 8. The GDFP-tNA compkx was bound to CTLL for 1 hour at 4 degrees C in binding medium as described in Example 10. CTLL were then washed three times in ice cold PBS, and the final cell peiiet was , ' ' in a Tris buffer containing 150mM NaCI, 5mM EDTA, 0.02%
Na-azide, and 0.5~; Triton X-100 to gentiy Iyse the cells. The cell Iysate was spun briefly, the ~ was brought to 0.4N NaOH, and the sample was denatured at 60 degrees C for 1 hour. The sample was then applied Yia slot-blot onto G~n,~.~r~n Plus membrane (NEN). The blot was screered for the presence of the target plasmid by h~i ii~ iun to a p2P]-labeled CAT probe. The membrane was washed, and the signai from cell associated plasmid was quantified by a 1' ~' _ (Molecular Dynamics). Figure 11 shows association of plasmid to CTLL mediated by either theGAL4/lL-2 GDFP or a negative control leti~uiu.,Jt., Iysate designated "Bg." The binding assay showed the ability of the GAL4/lL-2 GDFP to mediate binding of plasmid tNA to CTLL.
Utiiitv The gene delivery fusion proteins of the present invention are useful increating non-viral gene delivery systems for delivering a p~ i.i., to a target cell.
_ _ _ _ _ _ . _ _ _ _ . _ . . .. _ . . _ _ _ _

Claims (36)

Claims

1. A fusion protein useful in delivering a targeted nucleic acid to a target cell, comprising a gene delivery fusion protein (GDFP), said GDFP comprising a nucleicacid binding domain (NBD) that binds to the targeted nucleic acid, fused to a gene delivery domain (GDD) that mediates delivery of the targeted nucleic acid to the target cell.

2. A fusion protein according to claim 1, wherein the targeted nucleic acid is a double-stranded nucleic acid.

3. A fusion protein according to claim 1, wherein the targeted nucleic acid is a single-stranded nucleic acid.

4. A fusion protein according to claim 1, wherein the targeted nucleic acid isDNA or an analog thereof.

5. A fusion protein according to claim 1, wherein the targeted nucleic acid is RNA or an analog thereof.

6. A fusion protein according to claim 1, wherein the targeted nucleic acid is in the form of a recombinant expression vector comprising a nucleotide sequence to be expressed in the target cell.

7. A fusion protein according to claim 6, wherein the nucleotide sequence to beexpressed is a nucleotide sequence that is not normally expressed in the target cell.

8. A fusion protein according to claim 6, wherein the nucleotide sequence to beexpressed is an antisense copy of a nucleotide sequence present in the target cell.

9. A fusion protein according to claim 1, wherein said GDFP further comprises a flexible polypeptide linker sequence ("flexon") between said nucleic acid binding domain and said gene delivery domain or within one of said domains.

10. A fusion protein according to claim 1, wherein said NBD comprises a nucleic acid binding component of a sequence-specific nucleic acid binding protein.

11. A fusion protein according to claim 1, wherein said NBD comprises a nucleic acid binding component of a sequence-non-specific nucleic acid binding protein.

12. A fusion protein according to claim 1, wherein said NBD comprises a multiplicity of nucleic acid binding (NB) components that bind one or more targeted nucleic acids.

13. A fusion protein according to claim 12, wherein said NBD comprises at least two NB components having differing binding specificities.

14. A fusion protein according to claim 12, wherein the NBD comprises a first NB component capable of binding to a specific cognate recognition sequence present in the targeted nucleic acid and a second NB component capable of binding non-specifically to the targeted nucleic acid.

15. A fusion protein according to claim 1, wherein the NBD further comprises a component capable of mediating condensation and/or charge neutralization of the targeted nucleic acid.

16. A fusion protein according to claim 1, wherein said gene delivery domain (GDD) comprises one or more components that facilitate delivery of a targeted nucleic acid to a target cell.

17. A fusion protein according to claim 16, wherein said components that facilitate delivery of a targeted nucleic acid to a target cell are selected from the group consisting of a binding/targeting component a membrane-disrupting component, a transport/localization component and a replicon integration component.

18. A fusion protein according to claim 16, wherein said GDD comprises two or more components that facilitate delivery of a targeted nucleic acid to a target cell, said components selected from the group consisting of a binding/targeting component, a membrane-disrupting component, a transport/localization component and a repliconintegration component.

19. A fusion protein according to claim 16, wherein said GDD comprises a binding/targeting component.

20. A fusion protein according to claim 16, wherein said GDD comprises a membrane disrupting component.

21. A fusion protein according to claim 16, wherein said GDD comprises a transport/localization component.

22. A fusion protein according to claim 16, wherein said GDD comprises a replicon integration component.

23. A fusion protein according to claim 22, wherein said replicon integration component is an integrase enzyme or a derivative thereof that retains integrase activity.

24. A macromolecular complex useful in delivering a targeted nucleic acid to a target cell, comprising a gene delivery fusion protein (GDFP) of claim 1 in association with a targeted nucleic acid.

25. A macromolecular complex according to claim 24, wherein said GDFP
comprises a replicon integration component.

26. A macromolecular complex according to claim 25, wherein said replicon integration component comprises a recombinase enzyme or a derivative thereof that retains recombinase activity, and wherein the targeted nucleic acid comprises NBD
cognate recognition sequences in proximity to terminal recombinase recognition sequences.

27. A macromolecular complex according to claim 26, wherein said recombinase is an integrase enzyme or a derivative thereof that retains integrase activity, and wherein the targeted nucleic acid comprises NBD cognate recognition sequences in proximity to terminal integrase recognition sequences.

28. A recombinant polynucleotide useful for preparing a gene delivery fusion protein, said polynucleotide comprising a coding sequence that encodes a GDFP ofclaim 1.

29. The recombinant polynucleotide of claim 28, wherein said polynucleotide is in the form of an expression vector comprising a transcriptional control region operably linked to said coding sequence.

30. A cell useful in preparing a gene delivery fusion protein, said cell containing an expression vector of claim 29.

31. A method of using a recombinant of claim 29 to produce a GDFP, said method comprising the steps of causing the recombinant polynucleotide to be transcribed and translated, and recovering a GDFP.

32. A method of using a GDFP of claim 1 to deliver said targeted nucleic acid to a target cell, the method comprising the steps of contacting the GDFP with the targeted nucleic acid to produce a GDFP/nucleic acid complex and contacting saidGDFP/nucleic acid complex with the target cell.

33. A cell produced by the method of claim 32 and progeny thereof.

34. The cell of claim 33 wherein the targeted nucleic acid is expressed in the cell as an RNA molecule selected from the group consisting of an RNA transcript, an antisense RNA, an RNA decoy and a ribozyme.

35. A method of using a GDFP of claim 23 to deliver said targeted nucleic acid to a target cell, the method comprising the steps of contacting the GDFP with the targeted nucleic acid to produce a GDFP/nucleic acid complex and contacting saidGDFP/nucleic acid complex with the target cell.

36. A cell produced by the method of claim 35 and progeny thereof, said cell comprising an integrated copy of said targeted nucleic acid.