Jeffrey S. Bartlett

Infectious Entry Pathway of Adeno-Associated Virus and Adeno-Associated Virus Vectors

ABSTRACT We have investigated the infectious entry pathway of adeno-associated virus (AAV) and recombinant AAV vectors by assessing AAV-mediated gene transfer and by covalently conjugating fluorophores to AAV and monitoring entry by fluorescence microscopy. We examined AAV entry in HeLa cells and in HeLa cell lines which inducibly expressed a dominant interfering mutant of dynamin. The data demonstrate that AAV internalizes rapidly by standard receptor-mediated endocytosis from clathrin-coated pits (half-time <10 min). The lysosomotropic agents ammonium chloride and bafilomycin A1 prevent AAV-mediated gene transfer when present during the first 30 min after the onset of endocytosis, indicating that AAV escapes from early endosomes yet requires an acidic environment for penetration into the cytosol. Following release from the endosome, AAV rapidly moves to the cell nucleus and accumulates perinuclearly beginning within 30 min after the onset of endocytosis. We present data indicating that escape of AAV from the endosome and trafficking of viral particles to the nucleus are unaffected by the presence of adenovirus, the primary helper virus for a productive AAV infection. Within 2 h, viral particles could be detected within the cell nucleus, suggesting that AAV enters the nucleus prior to uncoating. Interestingly, the majority of the intracellular virus particles remain in a stable perinuclear compartment even though gene expression from nuclear AAV genomes can be detected. This suggests that the process of nuclear entry is rate limiting or that AAV entry involves multiple pathways. Nevertheless, these data establish specific points in the AAV infectious entry process and have allowed the generation of a model for future expansion to specific cell types and AAV vector analysis in vivo.

Differential Activation of Innate Immune Responses by Adenovirus and Adeno-Associated Virus Vectors

ABSTRACT Adenovirus vectors induce acute inflammation of infected tissues due to activation of the innate immune system and expression of numerous chemokines and cytokines in transduced target cells. In contrast, adeno-associated virus (AAV) vectors are not associated with significant inflammation experimentally or clinically. We tested the ability of AAV vectors to induce the expression of chemokines in vitro and to activate the innate immune system in vivo. In human HeLa cells and murine renal epithelium-derived cells (REC cells) the adenovirus vector AdlacZ induced the expression of multiple inflammatory chemokines including RANTES, interferon-inducible protein 10 (IP-10), interleukin-8 (IL-8), MIP-1β, and MIP-2 in a dose-dependent manner. The use of AAVlacZ did not induce the expression of these chemokines above baseline levels despite 40-fold-greater titers than AdlacZ and greater amounts of intracellular AAVlacZ genomes according to Southern and slot blot analysis. This finding confirmed that the lack of AAVlacZ induction of chemokine was not due to reduced transduction. In DBA/2 mice, the intravenous administration of 2.5 × 1011 particles of AAVlacZ resulted in the rapid induction of liver tumor necrosis factor alpha (TNF-α), RANTES, IP-10, MIP-1β, MCP-1, and MIP-2 mRNAs. However, 6 h following injection, chemokine mRNA levels returned to baseline. As expected, administration of 10-fold less AdlacZ caused an induction of liver TNF-α and chemokine mRNAs that persisted for more than 24 h posttransduction. Whereas intravenous administration of 2.5 × 1011 particles of AAVlacZ triggered a transient infiltration of neutrophils and CD11b+ cells into liver, this response stood in contrast to widespread inflammation and toxicity induced by AdlacZ. Kupffer cell depletion abolished AAVlacZ but not AdlacZ-induced chemokine expression and neutrophil infiltration. In summary, these results show that AAV vectors activate the innate immune system to a lesser extent than do adenovirus vectors and offer a possible explanation for the reduced inflammatory properties of AAV compared to adenovirus vectors.

Insertional Mutagenesis of the Adeno-Associated Virus Type 2 (AAV2) Capsid Gene and Generation of AAV2 Vectors Targeted to Alternative Cell-Surface Receptors

Recombinant adeno-associated virus (AAV) vectors are of interest in the context of gene therapy because of their ability to mediate efficient transfer and stable expression of therapeutic genes in a wide variety of tissues. However, AAV-mediated gene delivery to specific cell populations is often precluded by the widespread distribution of heparan sulfate proteoglycan (HSPG), the primary cellular receptor for the virus. Conversely, an increasing number of cell types are being identified that do not express HSPG and are therefore poor targets for AAV-mediated gene transfer. To address these issues, we have developed strategies to physically modify AAV vectors and allow efficient, HSPG-independent, receptor-targeted infection. We began by generating a series of 38 virus capsid mutants containing peptide insertions at 25 unique sites within the AAV capsid protein. The mutant viruses were characterized on the basis of their phenotypes and grouped into three classes: class I mutants (4 of 38) did not assemble particles; class II mutants (14 of 38) assembled noninfectious particles; and class III mutants (20 of 38) assembled fully infectious particles. We examined the HSPG-binding characteristics of the class II mutants and showed that a defect in receptor binding was a common reason for their lack of infectivity. The display of foreign peptide epitopes on the surface of the mutant AAV particles was found to be highly dependent on the inclusion of appropriate scaffolding sequences. Optimal scaffolding sequences and five preferred sites for the insertion of targeting peptide epitopes were identified. These sites are located within each of the three AAV capsid proteins, and thus display inserted epitopes 3, 6, or 60 times per vector particle. Modified AAV vectors displaying a 15-amino acid peptide, which binds to the human luteinizing hormone receptor (LH-R), were generated and assessed for their ability to target gene delivery to receptor-bearing cell lines. Titers of these mutant vectors were essentially the same as wild-type vector. The LH-R-targeted vector was able to transduce ovarian cancer cells (OVCAR-3) in an HSPG-independent manner. Furthermore, transduction was shown to proceed via the LH-R and therefore treatment of OVCAR-3 cells with progesterone, to increase LH-R expression, accordingly increased LH mutant-mediated gene transfer. This technology may have a significant impact on the use of AAV vectors for human gene therapy.

RGD inclusion in VP3 provides adeno-associated virus type 2 (AAV2)-based vectors with a heparan sulfate-independent cell entry mechanism

Recombinant adeno-associated virus (AAV) has become an attractive vector system for a number of gene therapy paradigms. However, the utility of AAV vectors is often limited by the absence of heparan sulfate proteoglycan (HSPG), the viruss primary attachment receptor, on the desired target cell population. In order to achieve HSPG-independent gene delivery, several groups have shown that the endogenous tropism of AAV can be expand by genetically altering the viral capsid. However, the parameters of this developing technology have yet to be defined and it has not yet been determined if these modified vectors actually infect cells via these engineered interactions. Previously we constructed a series of insertion mutants spanning the AAV capsid protein gene and identified specific sites that can tolerate the insertion of small exogenous peptides. Here we describe a number of sites within the AAV capsid gene that can be used for the insertion of integrin-targeting peptide epitopes. Incorporation of an Arg-Gly-Asp (RGD)-containing peptide at these sites enables AAV to infect integrin-expressing cells independent of HSPG. Mutant AAV vectors displaying these peptide ligands can be produced to wild-type titer and have been shown to specifically interact with the targeted integrin receptors and mediate infection via this interaction. We report significant increases in gene transfer to Raji, K562, and SKOV-3 cell lines that express integrin, but little HSPG, suggesting that rAAV vectors displaying RGD peptides may be of great utility for treatment of neoplasms characterized by the deficiency of HSPG expression. We have also demonstrated that due to their expanded tropism, these novel vectors are capable of efficient transduction of AAV2-resistant tumors in vivo suggesting that they may offer significant therapeutic advantages.

Molecular Characterization of Adeno-Associated Viruses Infecting Children

ABSTRACT Although adeno-associated virus (AAV) infection is common in humans, the biology of natural infection is poorly understood. Since it is likely that many primary AAV infections occur during childhood, we set out to characterize the frequency and complexity of circulating AAV isolates in fresh and archived frozen human pediatric tissues. Total cellular DNA was isolated from 175 tissue samples including freshly collected tonsils (n = 101) and archived frozen samples representing spleen (n = 21), lung (n = 16), muscle (n = 15), liver (n = 19), and heart (n = 3). Samples were screened for the presence of AAV and adenovirus sequences by PCR using degenerate primers. AAV DNA was detected in 7 of 101 (7%) tonsil samples and two of 74 other tissues (one spleen and one lung). Adenovirus sequences were identified in 19 of 101 tonsils (19%), but not in any other tissues. Complete capsid gene sequences were recovered from all nine AAV-positive tissues. Sequence analyses showed that eight of the capsid sequences were AAV2-like (∼98% amino acid identity), while the single spleen isolate was intermediate between serotypes 2 and 3. Comparison to the available AAV2 crystal structure revealed that the majority of the amino acid substitutions mapped to surface-exposed hypervariable domains. To further characterize the AAV capsid structure in these samples, we used a novel linear rolling-circle amplification method to amplify episomal AAV DNA and isolate infectious molecular clones from several human tissues. Serotype 2-like viruses were generated from these DNA clones and interestingly, failed to bind to a heparin sulfate column. Inspection of the capsid sequence from these two clones (and the other six AAV2-like isolates) revealed that they lacked arginine residues at positions 585 and 588 of the capsid protein, which are thought to be essential for interaction with the heparin sulfate proteoglycan coreceptor. These data provide a framework with which to explore wild-type AAV persistence in vivo and provide additional tools to further define the biodistribution and form of AAV in human tissues.

Osteogenic gene regulation and relative acceleration of healing by adenoviral‐mediated transfer of human BMP‐2 or ‐6 in equine osteotomy and ostectomy models

This study evaluated healing of equine metatarsal osteotomies and ostectomies in response to percutaneous injection of adenoviral (Ad) bone morphogenetic protein (BMP)‐2, Ad‐BMP‐6, or beta‐galactosidase protein vector control (Ad‐LacZ) administered 14 days after surgery. Radiographic and quantitative computed tomographic assessment of bone formation indicated greater and earlier mineralized callus in both the osteotomies and ostectomies of the metatarsi injected with Ad‐BMP‐2 or Ad‐BMP‐6. Peak torque to failure and torsional stiffness were greater in osteotomies treated with Ad‐BMP‐2 than Ad‐BMP‐6, and both Ad‐BMP‐2‐ and Ad‐BMP‐6‐treated osteotomies were greater than Ad‐LacZ or untreated osteotomies. Gene expression of ostectomy mineralized callus 8 weeks after surgery indicated upregulation of genes related to osteogenesis compared to intact metatarsal bone. Expression of transforming growth factor beta‐1, cathepsin H, and gelsolin‐like capping protein were greater in Ad‐BMP‐2‐ and Ad‐BMP‐6‐treated callus compared to Ad‐LacZ‐treated or untreated callus. Evidence of tissue biodistribution of adenovirus in distant organs was not identified by quantitative PCR, despite increased serum antiadenoviral vector antibody. This study demonstrated a greater relative potency of Ad‐BMP‐2 over Ad‐BMP‐6 in accelerating osteotomy healing when administered in this regimen, although both genes were effective at increasing bone at both osteotomy and ostectomy sites.

Mosaic vectors comprised of modified AAV1 capsid proteins for efficient vector purification and targeting to vascular endothelial cells

Vascular-targeted gene therapies have the potential to treat many of the leading causes of mortality in the western world. Unfortunately, these therapies have been ineffective due to poor vascular gene transfer. The use of alternative virus serotypes and the incorporation of vascular targeting ligands into vectors has resulted in only modest increases in vascular gene transfer. Adeno-associated virus (AAV) 1 has shown the most promise among the AAV vectors for the transduction of vascular endothelial cells. However, no straightforward small-scale purification strategy exists for AAV1 as it does for AAV2 making it difficult to quickly produce AAV1 vector for analysis. Here we have combined two AAV1 capsid protein modifications to enhance vascular gene transfer and allow easy purification of vector particles. Mosaic vector particles have been produced comprised of capsid proteins containing the well-characterized RGD4C modification to target integrins present on the vasculature, and capsid proteins containing a modification that permits metabolic biotinylation and efficient purification of mosaic particles by avidin affinity chromatography. We show that the RGD modification results in a 50–100-fold enhancement in endothelial cell gene transfer that is maintained in biotinylated mosaic AAV1 particles. These results suggest that mosaic virions hold significant promise for targeted gene delivery to the vasculature.

Complement Is an Essential Component of the Immune Response to Adeno-Associated Virus Vectors

ABSTRACT Adeno-associated virus (AAV) vectors are associated with relatively mild host immune responses in vivo. Although AAV induces very weak innate immune responses, neutralizing antibodies against the vector capsid and transgene still occur. To understand further the basis of the antiviral immune response to AAV vectors, studies were performed to characterize AAV interactions with macrophages. Primary mouse macrophages and human THP-1 cells transduced in vitro using an AAV serotype 2 (AAV2) vector encoding green fluorescent protein did not result in measurable transgene expression. An assessment of internalized vector genomes showed that AAV2 vector uptake was enhanced in the presence of normal but not heat-inactivated or C3-depleted mouse/human serum. Enhanced uptake in the presence of serum coincided with increased macrophage activation as determined by the expression of NF-κB-dependent genes such as macrophage inflammatory protein 2 (MIP-2), interleukin-1β (IL-1β), IL-8, and MIP-1β. AAV vector serotypes 1 and 8 also activated human and mouse macrophages in a serum-dependent manner. Immunoprecipitation studies demonstrated the binding of iC3b complement protein to the AAV2 capsid in human serum. AAV2 did not activate the alternative pathway of the complement cascade and lacked cofactor activity for factor I-mediated degradation of C3b to iC3b. Instead, our results suggest that the AAV capsid also binds complement regulatory protein factor H. In vivo, complement receptor 1/2- and C3-deficient mice displayed impaired humoral immunity against AAV2 vectors, with a delay in antibody development and significantly lower neutralizing antibody titers. These results show that the complement system is an essential component of the host immune response to AAV.

Site-specific modification of AAV vector particles with biophysical probes and targeting ligands using biotin ligase.

We have developed a highly specific and robust new method for labeling adeno-associated virus (AAV) vector particles with either biophysical probes or targeting ligands. Our approach uses the Escherichia coli enzyme biotin ligase (BirA), which ligates biotin to a 15-amino-acid biotin acceptor peptide (BAP) in a sequence-specific manner. In this study we demonstrate that by using a ketone isotere of biotin as a cofactor we can ligate this probe to BAP-modified AAV capsids. Because ketones are absent from AAV, BAP-modified AAV particles can be tagged with the ketone probe and then specifically conjugated to hydrazide- or hydroxylamine-functionalized molecules. We demonstrate this two-stage modification methodology in the context of a mammalian cell lysate for the labeling of AAV vector particles with various fluorophores, and for the attachment of a synthetic cyclic arginine-glycine-aspartate (RGD) peptide (c(RGDfC)) to target integrin receptors that are present on neovasculature. Fluorophore labeling allowed the straightforward determination of intracellular particle distribution. Ligand conjugation mediated a significant increase in the transduction of endothelial cells in vitro, and permitted the intravascular targeting of AAV vectors to tumor-associated vasculature in vivo. These results suggest that this approach holds significant promise for future studies aimed at understanding and modifying AAV vector-cellular interactions.

Engineering Cellular Resistance to HIV-1 Infection In Vivo Using a Dual Therapeutic Lentiviral Vector

We described earlier a dual-combination anti-HIV type 1 (HIV-1) lentiviral vector (LVsh5/C46) that downregulates CCR5 expression of transduced cells via RNAi and inhibits HIV-1 fusion via cell surface expression of cell membrane-anchored C46 antiviral peptide. This combinatorial approach has two points of inhibition for R5-tropic HIV-1 and is also active against X4-tropic HIV-1. Here, we utilize the humanized bone marrow, liver, thymus (BLT) mouse model to characterize the in vivo efficacy of LVsh5/C46 (Cal-1) vector to engineer cellular resistance to HIV-1 pathogenesis. Human CD34+ hematopoietic stem/progenitor cells (HSPC) either nonmodified or transduced with LVsh5/C46 vector were transplanted to generate control and treatment groups, respectively. Control and experimental groups displayed similar engraftment and multilineage hematopoietic differentiation that included robust CD4+ T-cell development. Splenocytes isolated from the treatment group were resistant to both R5- and X4-tropic HIV-1 during ex vivo challenge experiments. Treatment group animals challenged with R5-tropic HIV-1 displayed significant protection of CD4+ T-cells and reduced viral load within peripheral blood and lymphoid tissues up to 14 weeks postinfection. Gene-marking and transgene expression were confirmed stable at 26 weeks post-transplantation. These data strongly support the use of LVsh5/C46 lentiviral vector in gene and cell therapeutic applications for inhibition of HIV-1 infection.