gene therapy protocols

Met hods for Gene Transfer Using DNA-Adenovirus Conjugates David T. Curie1 1. Introduction Strategies have been developed to accomphsh gene delivery via the recep- tor-mediated pathway employing molecular conjugate vectors (Z-13). As cells possess endogenous pathways for internalization of macromolecules, the utili- zation of these pathways for the purpose of DNA delivery represents a strategy that potentially allows certain practical advantages. In this regard, these cellu- lar internalization pathways can be highly efficient. For example, internaliza- tion of the iron transport protein transferrin can be on the order of thousands of molecules per minute per cell (1415,. These pathways thus represent a poten- tially efficient physiologic method to transport DNA across the cell membrane of eukaryotic cells. To accomplish gene transfer via receptor-mediated endocy- tosis, a vehicle must be derived that allows DNA entry into these cellular path- ways. For this purpose, molecular conjugate vectors have been derived. These vector agents consist of two linked functional domains: a DNA-binding domain to transport the DNA as part of the vector complex, and a ligand domain to target a cellular receptor that allows entry of the conjugate-DNA complex into a receptor-mediated endocytosis pathway. For incorporating DNA into the complex for gene delivery, binding must be achieved in a nondamaging, reversible manner. For this linkage, an electrostatic association between the binding domain and the nucleic acid is accomplished. To achieve this, the DNA binding domain is comprised of a polycationic amine, such as poly(L)lysine. This can associate with the negatively charged DNA in an electrostatic, noncovalent manner. To achieve entry of the complex through a receptor-medi- ated pathway, a ligand for the target cell is utilized. The ligand domain is covalently linked to the polylysine to create the molecular conjugate vector. From Methods m Molecular Medmne, Gene Therapy Protocols Edited by. P Robbms Humana Press Inc , Totowa, NJ 1 2 Curie1 The ligand domain may be a native or synthetic cell surface receptor hgand, an anttreceptor antibody, or other agent that allows specific association with tar- get cell membranes. The interaction of the DNA with its binding domain serves not only to attach it to the molecular conjugate vector, but also to condense it into a compact circular toroid (5). In this configuration, the llgand domain is pre- sented on the exterior of the complex. By virtue of its surface location, the ligand is free to recognize its target receptor on the cell surface membrane. Thus, after binding by means of the ligand domain, the conjugate-DNA com- plex IS internalized by the receptor-mediated pathway. In this schema, the initial localization after internalization is within the cellular endosome. The conjugate-DNA complex may then achieve DNA delivery to the target cell nucleus, where expression of the transported heterologous sequences may occur. Alternatively, the complex may traffic to lysosomal pathways, which would result in degradation of the conjugate-DNA complex. Thus, by exploit- mg an endogenous cellular entry pathway, the conjugate-DNA complex achieves target cell internalization. After internahzation, the complex can be subject to multiple possible fates; however, optimally, DNA delivery to the nucleus permits heterologous gene expression. The delivery of genes by the receptor-mediated pathway offers certain unique features and potentials (4,16). Because the system is synthetic, the capacity exists to prepare large amounts of the conjugate vector. As delivery is by a physiologic cellular pathway, toxicity associated with membrane pertur- bation is circumvented. Thus, the potential exists to admimster the vector on a repetitive, or continuous basis. Importantly, the marked plasticity of the system allows the potential to derive a vector with the properties of cell-specific tar- geting. That is, through choice of the hgand domain, it is possible to selec- tively target cells possessing receptors for that ligand. This would potentially allow employment for those gene therapy applications requiring cell-specific delivery of therapeutic genes. Finally, the molecular conjugate vector system is devoid of viral gene elements. It would thus be devoid of the potential safety hazards deriving from the presence of viral gene sequences and functions. Gene delivery by the receptor-mediated endocytosis pathway was first described by Wu et al. (2). To selectively target hepatocytes, efforts were directed toward accomplishing cellular internalization through a receptor unique to this cell type. In this regard, hepatocytes possess unique receptors for recognition and clearance of asialoglycoproteins. This receptor is a constituent of a high efficiency internalization pathway specific to hepatocytes. To target through this receptor, asialoorosomucoid, a physiologic ligand for this recep- tor, was chemically conjugated to poly(L)lysine. The resulting conjugates could form complexes with DNA that could be shown to accomplish gene delivery DNA-Adenovirus Conjugates Tf 3 l Binding Target cell I Fig. 1. Gene transfer via the receptor-mediated endocytosis pathway. A bifunc- tional molecular conjugate is employed to bind DNA and transport it via cellular mac- romolecular transport mechanisms. The molecular conjugate \:ector consists of a DNA-binding domain, comprised by a cationic polylysine moiety that is co\,alently linked to a ligand for a cell surface receptor, in this case transferrin. Plasmid DNA bound to the polylysine moiety of the conjugate undergoes marked condensation to yield an 80- to 1 00-nm toroid with surface-localized transferrin molecules. When the transferrin ligand domain is bound by its corresponding cell surface receptor, the con- jugate is internalized by the receptor-mediated endocytosis pathway, cotransporting bound DNA. Escape from the cell vesicle system is achieved by a fraction of the inter- nalized conjugate-DNA complex to achieve nuclear localization where heterologous gene expression occurs. specifically through the asiologlycoprotein receptor of hepatocytes. Utilizing this schema, it was shown that selective delivery to hepatocytes could be accomplished both in vitro as well as after in vivo delivery of the conjugates (1,2,17). Utilizing a similar strategy, Birnstiel et al. developed a molecular conjugate system for achieving DNA delivery through the transferrin internal- ization pathway (Fig. 1; refs. 3,7). In this context, transferrin is internalized by a receptor-mediated endocytosis mechanism as part of a recycling pathway (1.5). The transferrin receptor is expressed in a ubiquitous manner but is rela- tively enriched in proliferative cells and cells of the hematopoietic derivation (14). It was demonstrated that gene transfer could also be accomplished through the transferrm internalization pathway (Fig. 1). In addition to these molecular conjugate vector strategies, gene delivery via the receptor-medi- ated endocytosis pathway has been described utilizing antibody against the polymerized IgA receptor (11) as well as surfactant protein C (18) as the ligand domain of the vector. Thus, the receptor-mediated schema of gene delivery has been developed for an increasing number of gene transfer appli- cations to capitalize on the potential to achieve nontoxic cellular entry as well as cell-specific gene delivery. Despite the many potential advantages of receptor-mediated gene delivery, the efficacy of molecular conjugate vectors, m practice, has been idiosyncratic (4,8). For many target cells, despite the presence of the requisite cell surface receptor, gene transfer via the corresponding internalization pathway has not resulted in efficient expression of transferred DNA. Analysis of the cellular uptake of conjugate-DNA complexes in these mstances has frequently demon- strated effective cellular internalization of the conjugate-DNA complex (3). In this regard, the internalized complexes may be demonstrated within cellular endosomes. This findmg suggests that the Inefficient gene expression 1s not related to initial binding and internalization steps, but reflects events occurrmg after cellular entry of the DNA-conlugate complex. Thus, in some instances, the conjugate-DNA complex may be entrapped withm cellular endosomes and the DNA thus cannot access the nucleus. Consistent with this concept, it has been reported that treatment of cells with selected lysosomotropic agents can significantly augment gene expression in conjugate-transfected cells (81. This is consistent with the fact that loss of conjugate-DNA complexes through cel- lular degradative processes may be etiologic of their inability to achieve nuclear localizatton and thus expression of transferred genes. Taken together, these findings illustrate a fundamental flaw of receptor-mediated gene transfer strat- egy as initially conceptualized: Despite the fact that the molecular conjugate vector possesses an efficient mechanism to achieve cellular internalization, the fact that it lacks a specific mechanism to escape entrapment within the endosome limits effective gene transfer and expression. This suggests that if the molecular conjugate vector possessed an endosome escape mechanism, the internalized DNA could avoid targetmg to cellular pathways eventuating its destruction, with a favorable outcome on gene transfer efficiency. 1.1. Adenovirus Facilitation of Receptor-Mediated Gene Delivery In developing methods to overcome endosome entrapment of the conjugate- DNA complexes, consideration was given to the entry mechanism of certain viruses. For both enveloped and nonenveloped viruses, the first step to cellular DNA-Adenovirus Conjugates 5 entry involves binding to a specific cellular receptor. For a subset of these viruses, after binding, internalization occurs by virtue of the receptor-mediated endocytosis pathway (29,20). After cellular internalization, these vnuses then utilize specific mechanisms to allow endosome escape of their genetic material so that the viral life cycle may be completed in the cell nucleus or cytosol. The viral entry pathway thus possesses certain parallels to the entry pathway of molecular conjugate vectors; like the conjugate vector, viruses may possess specific and efficient receptor-mediated entry mechanisms. However, apart from the conjugate vector, after internalization, viruses possess spectfic mecha- nisms to achieve escape from entrapment within the cell vesicle system for completion of their infectious cycle. It was thus hypothesized that the endosome escape property of certain viruses could potentially be exploited to overcome endosome entrapment of the conjugate-DNA complex, and thus facilitate efficient gene transfer. In this regard, previous studies had lmked viral entry to enhanced cellular uptake of various macromolecules Carresco utilized both enveloped and nonenveloped viruses to show augmented cellular internalization of macromolecules (21). These studies did not delineate the mechanistic basis for this phenomenon, or distinguish between membrane- bound or fluid-phase molecules; however, the linkage between viral uptake and enhanced cellular entry of heterologous molecules was clearly established. Specific facilitation of hgands via the receptor-mediated pathway was demon- strated by Pastan et al. (22). This group has developed a system of antitumor therapeutics based on dehvery of ligand-toxin chimeras via receptor-mediated endocytosis. In these studies, rt was found that entrapment of the chimeric toxin molecule in the cellular endosome limited tumoricidal efficacy. It was noted in this instance that this limitation could be overcome by codelivery of adenovr- rus with the chimeric toxin. In this schema, the virus colocalized wrthin the same cellular endosome as the conjugate during mternalizatton. Further, rt could be shown that it was the adenovirus’ ability to disrupt cellular endosomes that allowed ingress of the conjugate into the cytosol, where its activity was thus potentiated. Thus, this work established that adenovirus enters cells via receptor-mediated endocytosis, and during this process, it may colocahze to cellular endosomes with other receptor-bound llgands. The virus exits the endosome vta a membrane disruption step that may also allow egress of other endosome contents. From the standpoint of exploiting this capacity of the adenovirus to achieve endosome escape, it is noteworthy that this effect is mediated by viral capsid proteins and independent of viral gene expression. In this regard, the entry cycle of the adenovnus has been partially elucidated (19,23,24). The virus first achieves target cell attachment through binding to a specific cell surface recep- tor. Whereas this receptor has not been characterized, its presence has been established by functional studies and it has been shown to be expressed m a rather ubiquitous manner. The adenovn-us accomplishes this initial binding step by virtue of a specific capsid protem designed fiber. After binding to the cellu- lar recognition receptor, uptake is facilitated by subsequent bmdmg of specific viral capsid regions to cellular proteins that act as uptake “triggers” (25). After this initial uptake step, the virion is then localized within cellular endosomes. After cellular localization within the endosome, the virus accomplishes endosome disruption to achieve escape from the cell vesicle system. Acidifica- tion of the endosome IS crucial to the ability of the vnus to achieve this vesicle disruption step. In this regard, cellular targets with defective acidification of their endosomes, or alternatrvely, treatment of normal cells with agents to aug- ment endosomal pH, both have the effect of hmitmg adenovnal propagation (26). Importantly, replication-defective adenoviral strains deleted m specific gene regions may still, nonetheless, complete the same prmcipal entry steps as wild-type viruses. It is hypothesized that acidification of the endosome induces alterations in the conformation of certain capsid proteins (19,261. This induces changes m then- hydrophobicity allowing them to thus interact with the endosome membrane m a manner to achieve vesicle disruption. Thus, it IS the capsid protems that medtate the effect of endosome disruption, viral gene expression is not an essential feature of this process. Based on this concept, it was hypothesized that the entry process of the adenovnus could be exploited to achteve endosome escape of the mternal- ized conjugate-DNA complex. In this schema, the provision of endosome disruption functions in tracts would be anticipated to augment the overall gene transfer efficiency mediated by the molecular conjugates (Fig. 2). To test this hypothesis, transferrin-polylysme-DNA complexes containing a tire- fly luctferase reporter plasmid DNA were codelivered to HeLa cells m con- Junction with the repltcation-defective adenovirus d1312. This use of the replication-defective adenovirus provided a convenient means to separate the possible effects mediated by viral entry and vtral gene expression, as this adenovirus strain in defective in its gene expression secondary to genomic deletions m early viral gene regions (27). It could be seen that with increas- ing input of adenovnus there was a corresponding increase in the level of luciferase gene expression detected (9). This adenovnal augmentation plateaued at a level of luciferase gene expression more than 2000-fold the levels observed when transfer&-polylysine conlugates were utilized without virus. Significantly, the amount of virus required to achieve these plateau levels of augmented gene expression corresponded to the number of receptors for the adenovnus on the target cell (28). Thus, the characteristics of receptor-mediated endocy- tosis facilitation by this route were saturable, as would be expected m a receptor-limiting context DNA-Adenovirus Conjugates 7 Fig. 2. Mechanism of adenoviral facilitation of molecular conjugate-mediated gene transfer. After binding to their respective cell surface receptors, cointernalization of the transferrin-polylysine conjugate and the adenovirus is within the same endocytotic vesicle. Adenovirus-mediated endosome disruption allows vesicle escape for both the virion and the conjugate-DNA complex. To determine the mechanistic basis of the virus’ ability to augment gene trans- fer mediated by molecular conjugates, steps were employed to uncouple virus entry and virus-mediated endosome disruption. In this regard, mild heat treat- ment of the adenovirus will selectively ablate the ability of the adenovirus to accomplish endosome disruption, without impairing its ability to bind to target cells (28). Additionally, this magnitude of heat treatment does not affect the struc- tural integrity of the adenoviral genome. Thus, by selectively ablating the viral endosome disruption capacity, its contribution to adenoviral-mediated augmen- tation of molecular conjugate gene transfer could be ascertained. In these experi- ments, heat treatment completely abrogated the ability of the virus to augment the conjugate’s gene transfer capacity. This finding establishes that it is specifi- cally the viral property of endosomolysis that contributes to its capacity to aug- ment the conjugate’s gene transfer capacity. This underscores the fact that it is the molecular conjugate vector’s lack of an endosome escape mechanism that repre- sents its principal limitation to achieving efficient gene transfer to target cells. 8 Curie1 The augmented levels of net gene expression accomplished by the adenovi- ral augmentation of molecular conjugates are consistent with either increased gene expression in a transfected cell subset or an increased number of cells transfected. To distmgulsh between these possibilities, HeLa cells were trans- fected with transferrm-polylysine conjugates containing a /3-galactosidase reporter gene (29). Cells transduced with the transferrin-polylysme-DNA com- plexes alone demonstrated a transduction frequency of cl%. When adenovirus was added as a facilitator, however, >90% of the HeLa cells showed expres- sion of the P-galactosidase gene. Thus, the adenovnal augmentation of conju- gate-mediated transfer allowed for a significantly enhanced frequency of transfection. To demonstrate further the level to whtch adenovirus could increase the efficiency of gene transfer mediated by conjugate-DNA com- plexes, limiting dilutions of transferrin-polylysine-luciferase DNA complexes were delivered to cells with or without adenovirus. It could be demonstrated that, in the presence of adenovirus, the same levels of heterologous gene expression were noted as when two orders of magnitude more DNA were delivered without adenoviral augmentation (9). Thus, the vnus appears to con- fer a high level of efficiency on the process of DNA delivery mediated by molecular conjugates. Importantly, the phenomenon of adenoviral augmenta- tion of conjugate-mediated delivery could be observed in a variety of cell types treated in this manner. In analyzing multiple different cellular targets, the free adenovirus stgmficantly augmented gene expression levels over levels seen with molecular conjugates alone. Additionally, certain cell types that appear refractory to transferrin-polylysme-mediated gene transfer demonstrate sus- ceptibility only m the presence of adenovirus (9). It IS likely that, in these instances, there was effective mternahzation of conjugate-DNA complex, but heterologous gene expression was absent secondary to more complete endosome entrapment of the conjugate-DNA complex. Thus, the susceptibtlity of these cells to conjugate-mediated gene transfer was only manifest after codelivery of adenovirus. The selective exploitation of adenoviral entry features is made possible because it is adenoviral capsid proteins that mediate the endosome disruption step of viral entry. In this context, viral gene expression is irrelevant to the ability of the adenovirus to facilitate molecular conjugate entry. Steps may thus be undertaken to ablate vnal gene elements and spare the capacity of the capsid to accomplish cell vesicle disruption. In this regard, it has been shown that ultraviolet (UV) light and UV light plus psoralen can be used to ablate viral infectivity and allow retention of the virus’ ability to facilitate molecular conjugate-mediated gene transfer (29). Thus, m this context, it is possible to render the adenoviral genome inactive m two complementing manners: genetic deletions of the adenoviral genome and physical inactivation of the adenoviral DNA-Adenovirus Conjugates 9 genome. This IS in marked contrast to recombinant vu-al vectors, whereby the integrity of viral gene elements is crucial, since the heterologous sequences are contained therein. Thus, for recombinant viral vectors, it 1s not possible to take steps such as UV treatment to more completely inactlvate viral gene ele- ments. An additional feature that derives from this strategy is the flexibility allowed in DNA delivery. In this regard, the polylysine component of the molecular conjugate interacts with DNA in a sequence-independent manner. Thus, DNA of any design can be incorporated mto the Conjugate-DNA com- plex and dehvered for purposes of gene transfer. Furthermore, the fact that the heterologous sequences are not incorporated into a viral genome minimizes the possibility of interactions among the distinct regulatory regions. Of addl- tional practical significance, because the heterologous DNA is not packaged into a virion capsid, the amount of DNA that may thus be delivered is not limited by the correspondmg packaging constraints. Using this approach, DNA plasmids of up to 48 kb have been delivered (29). Thus, an enhanced flexibility in terms of size and design of delivered DNA derives from this strat- egy of gene transfer. 1.2. Adenovirus-Component Molecular Conjugates for Receptor-Mediated Gene Delivery The utihty of the adenovirus in facilitating adenoviral entry wz trans sug- gests that it might also be possible to accomplish this with the adenovnus f%nc- tioning in cis. Thus, smce molecular conjugates were functionally limited by their lack of an endosome escape mechanism, and since adenovuus possessed such a mechanism, it seemed logical to incorporate the adenovirus into the structure of the molecular conjugate vector. The first technical barrier to achieving this construction was the attachment of the adenovirus to the polyl- ysine-DNA binding moiety. In attaching moieties to the adenoviral capsid, a potential complication undermining this strategy would have been perturba- tion of the capsid proteins involved in adenoviral binding and entry. In this regard, the adenoviral capsid proteins fiber and penton are thought to be of major importance to these entry steps (19,26). The hexon protein is thought to subserve the function of “scaffolding” of the capsid and is less important in viral entry processes. It was thus determined that the most propitious site to accomplish linkage was via the hexon protein. To achieve this, the strategy delineated in Fig. 3 was carried out. The hexon gene of the adenovirus was first isolated. Specific mutations were introduced into the gene sequence of the hexon gene to create a region coding for a heterologous epitope. The gene sequence altered corresponded to a region of the hexon protein known to be present in the exterior, surface face of the virus. Thus, by genetic techniques, it was possible to generate a chimeric adenovirus with the heterologous epitope IO Curie/ Attachment site Anti-Ml’1 Adenovirus genome 36kb t M.Pl sequence Fig. 3. Construction of chimeric adenovirus containing heterologous epitope in sur- face region of hexon capsid protein. Since the adenoviral capsid proteins fiber and penton are important mediators of the adenoviral entry mechanism, attachment of capsid-bound DNA was targeted to the hexon protein. A specific attachment site for an immunologic linkage was created by introducing a heterologous epitope into the surface region of the hexon protein by site-directed mutagenesis of the corresponding region of the adenoviral hexon gene. The introduced foreign epitope is a portion of Mycoplasma pneumoniae Pl protein. localized in a manner to permit nonneutralizing interaction with an MAb spe- cific for this epitope. After derivation of the chimeric adenovirus, an attach- ment schema could be carried out (Fig. 4). The antibody specific for the heterologous epitope served as the site of attachment of the polylysine-DNA- binding moiety. When condensed with DNA, the resulting toroid possessed surface localized immunoglobin capable of recognizing the heterologous epitope on the chimeric virus. The ability of the adenovirus-polylysine-DNA complexes to mediate gene transfer was evaluated using various components of the complete complex. It could be seen that the specific combination of epitope-marked virus, antibody- polylysine, and DNA resulted in a vector that was capable of achieving high levels of heterologous gene expression (13). In contrast, any other combina- tion of these components did not allow gene transfer to occur. As before, heat inactivation of the virion ablated the capacity of the complexes to accomplish gene transfer, indicating that it was the viral entry features that were respon- sible for the gene transfer capacity of the complex in this context as for the case of adenovirus functioning in trans. As an additional control, complexes were [...]... Curiel, D T (1994) Receptor-mediated gene delivery employing lectm-bmdmg specificity Gene Ther 1,255-260 40 Morgan, R A and Anderson, W F (1993) Human gene therapy Biochem 62, 191-217 41 Gao, L., Wagner, E., Cotten, M., Agarwal, S., Harris, C., Romer, M., Hu, P.-C., and Curiel, D (1993) Direct in vivo gene transfer to airway employing adenovnus-polylysine-DNA complexes Hum Gene Ther Ann Rev Miller, L., epithelium... DNA The adenovirus helper can be removed by a number of physical and genetic techniques Heating the virus lysate to 56°C for 30 min is one such strategy In this packaging system, one can generate helper-free stocks of AAV vectors at titers of 104-105/mL 1.6 Advantages and Disadvantages of rAA V Vectors for Gene Transfer and Gene Therapy As mentioned, several AAV vector systems have been developed that... enhance overall gene expression In addition to delivering large genes efficiently, it 1salso possible to deliver multiple DNA constructs simultaneously Despite their efficacy in vitro, use of molecular conjugates in vivo has been idiosyncratic Gao et al have shown that molecular conjugate vectors are able to mediate gene transfer in the airway epithelium of cotton rats (4Z) The observed gene transfer... molecules per cell resulted in levels of reporter gene expression detectable above background levels (13) This compares favorably to gene transfer mediated by other DNA-mediated gene transfer methods, where on the order of 500,000 DNA molecules per cell are required (30,31) Thus, the adenovirus-polylysine-DNA complexes are capable of extremely efficient gene transfer 12 Curie/ For lmkage of the polylysme-DNA-binding... and the vector psub201, containing a foreign gene inserted between the two AAV terminal repeats (in the presence of adenovirus), rescue, replication, and packaging of the foreign gene mto AAV particles occurs The adenovirus genome has been shown to activate the adenovirus terminal repeats onpAA V/Ad; this enhances the turning on of the AAV genes The rep gene products recognize the AAV czs-acting terminal... achieve gene delivery This strategy is thus distinct from the design of recombinant viral vectors In the instance of recombinant viral vectors, the overall entry mechanism of the virus is exploited to achieve gene delivery through the incorporation of the foreign gene mto the genome of the vn-us For the adenovn-us-component molecular conjugate vectors, viral entry features are exploited to facilitate gene. .. enhancer or promoter activity, and recombinant viruses generated using these elements function as vectors for stable transduction Expression of the gene or DNA sequence of choice in eukaryotrc cells is determined by the control of a transcriptional promoter included with the gene cassette(29,30) Recombinant AAV is among the newest of the possible genetic transfer vectors This once obscure virus possessesunique... profiles comparable to complexes employing immunologic methods of attachment When compared to the maximum levels of gene transfer achieved by free viral facilitation of molecular conjugate-mediated gene transfer, adenovirus m the linked configuration is capable of significantly higher levels of gene expression (data not shown) This likely reflects the different entry pathways of delivered DNA in the two... Austria References 1 Wu, G Y., Wilson, J M., Shalaby, F., Grossman, M., Shafritz, D A , and Wu, C H (1991) Receptor-mediated gene delivery in vivo Partial correction of genetic analbummemia m nagase rats J Biol, Chem 266, 14,33&14,342 2 Wu, G Y and Wu, C H (1988) Receptor-mediated gene delivery and expression zn vivo J Biol Chem 263, 14,621-14,624 3 Zenke, M., Steinlein, P., Wagner, E., Cotten, M., Beug,... Helm, M., and Chan, H (1989) Cattonic hposome mediated transfection Proc West Pharmacol Sot 32, 115-121 32 Cristiano, R J., Smith, L C., Kay, M A , Brinkley, B R., and Woo, S L (1993) Hepatic gene therapy: efficient gene delivery and expression in primary hepatocytes utilizing a conjugated adenovmrus-DNA complex Proc Natl Acad Sci USA 90, 11,548-l 1,552 33 Greber, U F., Willetts, M., Webster, P., and Helenms, . employment for those gene therapy applications requiring cell-specific delivery of therapeutic genes. Finally, the molecular conjugate vector system is devoid of viral gene elements. It would. hexon gene of the adenovirus was first isolated. Specific mutations were introduced into the gene sequence of the hexon gene to create a region coding for a heterologous epitope. The gene sequence. of gene delivery has been developed for an increasing number of gene transfer appli- cations to capitalize on the potential to achieve nontoxic cellular entry as well as cell-specific gene