The Proceedings of the American Thoracic Society 1:309-314 (2004)
© 2004 The American Thoracic Society
Adeno-associated Virus and Lentivirus Pseudotypes for Lung-directed Gene Therapy
James M. Wilson
Gene Therapy Program, Division of Medical Genetics, Department of Medicine, University of Pennsylvania Health System, and The Wistar Institute, Philadelphia, Pennsylvania
Correspondence and requests for reprints should be addressed to James M. Wilson, M.D., Ph.D., 204 Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104-4268. E-mail: wilsonjm{at}mail.med.upenn.edu
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ABSTRACT
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The enthusiasm for cystic fibrosis gene therapy that attended the initial cloning of the gene and in vitro correction of the genetic defect eventually diminished as we learned more about the limitations of vector technologies that were available in the 1980s and 1990s. Substantial progress has been made, however, over the last 5 years in developing second- and third-generation vector constructs that should be more useful in achieving gene transfer to the lung for the treatment of pulmonary diseases such as cystic fibrosis.
Key Words: recombinant • AAV • lentivirus • lung tropism
Isolation of the gene responsible for cystic fibrosis (CF) in 1989 ushered in a new era of research related to this inherited disease (1). The notion of gene therapy to cure CF captured the imagination of the community. Directing a normal version of the CF gene to the lung, where the clinical manifestations were the most problematic, became the goal of a number of investigators. Together with my colleagues, Francis Collins and Ray Frizzell, we demonstrated correction of the functional defect in an immortalized cell from a patient with CF through the use of a retroviral vector expressing the normal version of the CF gene (2). This provided additional evidence that the gene isolated through genetic linkage studies was indeed that responsible for the primary defect in CF. This brief review summarizes the progress made in developing a vector for CF gene therapy focusing on work from my laboratory. A number of important contributions have been made from other groups, although a thorough review of this literature is beyond the scope of this article.
It was initially believed, and later confirmed, that an important target for gene therapy in CF is the vast array of epithelial cells that line the conducting airways (3, 4). One attractive feature of gene therapy in this disorder is that the target cell population could potentially be accessed through a noninvasive route of delivery, i.e., inhalation. The retroviral vector used in the in vitro correction experiments, which was based on a murine leukemia virus, was studied for its ability to transduce (i.e., transfer and express a transgene) conducting airway epithelial cells after direct administration into the lungs of rodents. These experiments were disappointing in that very little gene transfer was accomplished (unpublished data). This was due in part to the fact that the murine leukemia–based retrovirus requires that the target cell be dividing for transduction to occur. Under normal conditions, the airway epithelia are relatively quiescent with respect to mitosis.
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ADENOVIRUS VECTORS
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Vectors based on recombinant adenoviruses were next evaluated for in vivo delivery of genes to airway epithelial cells. Human adenoviruses, in fact, are a frequent cause of upper respiratory infections. It was proposed, therefore, that they would be tropic for the airway epithelial cells and good gene delivery vehicles for CF. Recombinant forms of adenoviruses based on human serotype 5 were indeed available and had been characterized extensively as molecular tools for studying basic biological processes. One could disable the adenovirus by deleting an important immediate early gene (i.e., E1) and could incorporate into the vector genome minigene cassettes in excess of 6 kb. The E1 deleted adenoviruses were easily grown to high quantities through propagation in an available cell line called 293, which harbors a 5' region of the human Ad5 genome.
Delivery of human adenovirus 5 vectors into the airway of rodents yielded impressive results with high level gene transfer accomplished throughout the conducting airway and in alveolar cells (5). Similar efficiencies were achieved in larger animals including nonhuman primates (6). A number of limitations, however, were noted with adenoviral vectors in this application (see Figure 1 and Reference 7). Transgene expression was not stable, diminishing to undetectable levels within 2 to 4 weeks. Loss of transgene expression was associated with substantial inflammation in the areas of lung where vector-encoded transgene was expressed. Finally, transduction was not achieved when these animals were readministered a vector of the same serotype (8). Virtually all of the limitations of the adenovirus noted above could be ascribed to host-antigen–specific immune responses. As shown in Figure 1, inhibition of CD4 T cells using a depleting antibody produced a substantially different result with transgene expression persisting for at least 1 month, without inflammation, and with successful readministration of vector. We and others undertook a series of experiments to delineate the specific mechanisms of antigen specific immunity in this application. We learned that the adenovirus effectively activates cytotoxic T lymphocytes (CTLs) to vector transduced cells using the transgene, as well as the products of other viral genes as antigenic targets (7–9). The effector–CTL response extinguished transgene expression through elimination of the transduced cells and downregulation of transgene expression and contributed significantly to inflammation. The input adenoviral capsid proteins activated B lymphocytes, which secreted antibodies neutralizing the ability of the virus to infect, explaining the difficulties with readministration of vector (8).
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Figure 1. Immune responses to in vivo gene transfer with adenovirus vectors. C57BL/6 mice were instilled intratracheally with AdLacZ vectors (1011 particles) into the airway and subsequently necropsied for evaluation of LacZ expression in lung at Days 3 and 28. The animals were instilled intratracheally with 1011 particles of AdAlkPhos vector at Day 28, and 3 days later necropsied for histochemical analysis of AlkPhos expression. The animals were treated with and without a depleting antibody to CD4 T cells.
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Another limitation of the adenovirus system was the relative inefficiency by which gene transfer was achieved (10, 11). Despite the fact that this virus was felt to be tropic for the airway, in vivo gene transfer required high doses of the vector. It was subsequently learned that the primary receptor for human adenovirus 5, called the coxsackie adenovirus receptor (CAR), was localized to the basolateral surface of the conducting airway epithelial cells and therefore was not accessible to vector administered into the airway (12).
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RETROVIRUS VECTORS
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The use of retroviruses for in vivo gene delivery received a substantial boost with the development of vectors based on lentiviruses. The original retroviral vectors for gene therapy were based on endogenous murine viruses which, when developed as vectors, require that the target cell undergo cell division for transduction to be achieved. It was known that the human retrovirus, human immunodeficiency virus (HIV), was indeed capable of infecting nondividing cells such as macrophages and neurons. A number of vector systems based on this group of retroviruses, called lentiviruses, were developed. Naldini and coworkers created replication defective versions of HIV that were capable of transducing nondividing cells and achieving stable expression through integration of the provirus into chromosomal DNA (13). To broaden the tropism of the lentiviral vector, these investigators created chimeras using envelope glycoproteins from other viruses in a technique called pseudotyping (Figure 2). Using this strategy, they were able to decorate the surface of the lentiviral virion with the envelope glycoprotein from vesticular stomatitis virus (VSV-G). Indeed, the VSV-G HIV vector was shown to achieve efficient and stable transgene expression after in vivo gene transfer in a number of systems. However, this vector system was inefficient in terms of targeting differentiated airway epithelial cells via the apical surface (14). We proposed that this inefficiency is due to paucity of receptors for the VSV-G protein on the apical surface of conducting airway. We conducted a survey of surface glycoproteins from a number of unrelated viruses that were used to create HIV-vector pseudotypes (15). Representative families of the following viruses were included in this survey: rhabdovirus, oncoretrovirus, filovirus, orthomyxovirus, paramyxovirus, arenavirus, hep-DNAvirus, and coronavirus. The most dramatic results were obtained with HIV vector pseudotyped with the glycoprotein envelope from the Ebola Zaire (EboZ) filovirus. The wild-type version of the virus causes a lethal hemorrhagic fever. Figure 3 summarizes the results of mice instilled into the airway with solutions of either vehicle or an HIV-lentiviral vector expressing LacZ and pseudotyped with VSV-G or EboZ. High-level gene transfer was achieved throughout the conducting airway and cells lining submucosal glands with the EboZ pseudotyped vector. In recent studies, we localized domains within the EboZ envelope that confer high level gene transfer into the airway and have performed experiments in nonhuman primates (16).
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Figure 2. Pseudotyping retroviral vectors. 293 T cells are transfected with 3 vector plasmids: one expressing the transgene flanked by retroviral LTRs; another called the transfer vector, which contains gag-pol and other necessary accessory genes; and the envelope expresser, which is a plasmid expressing the envelope we wish to pseudotype, such as vesticular stomatitis virus (VSV-G).
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Figure 3. In vivo gene transfer of LacZ from HIV lentiviral vectors into C57BL/6 mice. Animals were instilled with solutions of lentiviral vector into the airway and subsequently analyzed for LacZ expression using histochemical characterization of lung tissue. This figure shows trachea harvested at Day 28 and Day 63, as well as lung harvested at Day 63. The three conditions are animals administered with vehicle, animals administered with VSV-G pseudotyped HIV-LacZ vector, and animals administered with EboZ pseudotyped HIV-LacZ vector. Reprinted by permission from Reference 15.
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ADENO-ASSOCIATED VIRUS VECTORS
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Another vector system under consideration for lung-directed gene transfer is based on the family of primate parvoviruses, called adeno-associated viruses (AAV). This family of viruses was originally isolated as contaminants in laboratory stocks of adenoviruses (Figure 4). A total of six different isolates of AAV were characterized over the last 40 years, five of which were from laboratory stocks of adenovirus, and one of which was isolated from a condylomatous wart. Antibodies generated against these various isolates suggested that most were serologically distinct and therefore have been called AAV serotypes 1–6. Serologic studies indicate that a substantial proportion of human populations have indeed been previously exposed to AAVs, although the exact nature of these natural infections, or their clinical sequelae, have not been defined (17). AAVs have a 4.9-kb single-stranded genome which, when replicated into a double-stranded form, expresses genes involved in replication (i.e., rep genes) and genes encoding proteins that form the capsid of the virus (i.e., cap genes, reviewed in Reference 18). In the absence of helper virus, AAV is capable of infecting cells and integrating its genome into chromosomal DNA in nondividing cells, which represents the latent phase of the virus life cycle and the one we wish to exploit in the setting of in vivo gene transfer. The AAV genome is propagated in the presence of helper virus, such as adenovirus, which is the strategy used in production of the vector. Replication defective versions of AAV serotype 2 were the first created and have undergone extensive testing in vivo in animals and humans.
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Figure 4. Electron micrograph of adenovirus prep contaminated with adeno-associated viruses (AAV). The large darkly stained particles are adenoviral virions, whereas the smaller lightly stained particles that surround many of the adenoviruses are AAV.
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In some tissues, AAV2 vectors are capable of transducing nondividing cells and conferring extraordinarily long expression of the transgene. The molecular state of the intracellular vector genome has not been well described, although it appears to exist both as integrated and nonintegrated forms (19, 20). The AAV vector is capable of introducing a transgene encoding a foreign protein without eliciting destructive CTL against the transduced cells (21). This has been extremely useful in avoiding host immune responses that could extinguish transgene expression. However, antibodies are indeed generated to the capsid proteins, which compromise readministration of the vector. Applications of in vivo gene therapy with AAV2 which have shown promise in animal models include gene transfer to the retina and skeletal muscle (22, 23). Vectors based on AAV2 have been evaluated for gene transfer to lung. Although some level of gene transfer is indeed detected in animal models, it is quite low (24). This is consistent with phase I studies of AAV2 vectors in subjects with CF where gene transfer in lung was detected at low levels with little direct evidence of transgene expression (25).
A number of the other known AAV serotypes were developed as vectors using a strategy similar to that described for the lentivirus-AAV pseudotypes in which the vector genome is derived from AAV2 while the packaging construct is a chimera between the rep gene from AAV serotype 2 and the cap gene from an alternative AAV serotype (Figure 5). The resulting vector contains an AAV2 based genome packaged in capsid from another AAV serotype. Figure 6 illustrates the performance of AAV pseudotypes expressing human alkaline phosphatase gene administered into the airway of C57BL/6 mice. This figure shows histochemical analysis of lung removed from animals 28 days after intranasal aerosol delivery of vectors based on AAV serotype 2 as well as serotype 1 (AAV2/1) and serotype 5 (AAV2/5) pseudotypes. Substantially higher levels of gene transfer are achieved with these pseudotypes as compared to AAV2 vectors. Specifically, AAV2/1 vectors showed efficient gene transfer in the conducting airway as well as alveolar cells, while AAV2/5 vectors showed high-level gene transfer in alveolar cells with less in conducting airway. The initial description of AAV5 pseudotypes for lung-directed gene transfer was from Zabner and colleagues (26).
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Figure 5. AAV pseudotypes. This figure depicts a strategy for creating AAV pseudotypes. The top line shows the two constructs used to produce AAV serotype vector in which the packaging plasmid has a rep and cap from AAV2 and the vector itself has a nugene flanked by AAV2 ITRs. The bottom line shows an AAV2 genome pseudotyped with a capsid from AAV1. The packaging construct now is a chimera using rep sequences from AAV2 and cap sequences from AAV1. The actual vector genome is AAV2 based.
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Figure 6. Analysis of AAV serotypes in mouse lung. C57BL/6 mice were delivered via intranasal aerosol 4.6 x 1011 genome copies of AAV vectors expressing alkaline phosphatase. Shown in this figure are animals that received AAV 2-, AAV2/1-, and AAV2/5-based vectors.
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We recently undertook a series of studies designed to characterize novel AAVs (27, 28). The goal of this work was to identify new AAV isolates with the hope that, when developed as pseudotypes, they would provide improved profiles of gene transfer and if isolated from nonhumans would not be neutralized by preexisting immunity in humans that occurs via natural AAV infections. We also hope to learn more about the biology of AAV from these studies. The strategy for pursuing these experiments is illustrated in Figure 7. Our goal was to use techniques of PCR to detect and recover AAV sequences in tissues that were left as latent proviruses following a preceding natural infection. Oligonucleotides were developed to highly conserved regions of the cap sequence separated by a variable region. Our studies initially focused on tissues from nonhuman primates. A typical PCR result is shown in Figure 8. DNA from a variety of tissues derived from rhesus macaques were analyzed using the PCR strategy described in Figure 7. As can be seen in Figure 8, this PCR action yielded detectable sequences in all animals diffusely throughout a number of tissues. We have now completed a similar kind of analysis in 259 tissues from 250 humans and 523 tissues from 214 nonhuman primates. Endogenous AAV sequences were detected in 20% of all nonhuman primate tissues and 18% of human tissues. Sequence analysis of the endogenous genomes revealed substantial heterogeneity primarily localized to the capsid genes. This heterogeneity of capsid sequence provides remarkable diversity of function, which is currently the subject of a number of studies.
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Figure 8. Survey of tissues from rhesus macaques for AAV signature sequences. DNA was extracted from the indicated tissues of rhesus macaques derived from either the Tulane or University of Pennsylvania (GTP) colonies. The DNAs were subjected to PCR using the strategy described in Figure 7, and the resulting reactions were fractionated on agarose gel. This slide summarizes the portion of the gel where the amplified 250–base pair fragment migrated.
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The availability of an expanded family of AAV sequences provides an opportunity to evaluate a number of new pseudotypes. We have in fact now sequenced and characterized over 100 novel AAV capsid structures, which are being incorporated into AAV pseudotypes as described in Figure 5 and evaluated as vectors for gene transfer into liver, muscle, and lung (Figure 9). In these studies, we use a secreted reporter gene, human alpha-1-antitrypsin, as a quantitative readout of in vivo gene transfer.
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FOOTNOTES
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This work was made possible through the generous support of the National Institutes of Health, the Cystic Fibrosis Foundation, and GlaxoSmithKline.
Conflict of Interest Statement: J.M.W. received $25,000 in 2003 as a one-time consulting fee for GenPhar and has patents licensed to Targeted Genetics Corp. (TGEN). The author previously held equity in Targeted Genetics, Corp.
The University of Pennsylvania has licensed gene transfer technology to various biological pharma companies.
(Received in original form September 7, 2004; accepted in final form September 21, 2004)
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ABSTRACT
ADENOVIRUS VECTORS
