Adeno-associated Virus Strains Research Articles

Integration of adeno-associated virus (AAV) into the genomes of most Thai and Mongolian liver cancer patients does not induce oncogenesis

BackgroundEngineered versions of adeno-associated virus (AAV) are commonly used in gene therapy but evidence revealing a potential oncogenic role of natural AAV in hepatocellular carcinoma (HCC) has raised concerns. The frequency of potentially oncogenic integrations has been reported in only a few populations. AAV infection and host genome integration in another type of liver cancer, cholangiocarcinoma (CCA), has been studied only in one cohort. All reported oncogenic AAV integrations in HCC come from strains resembling the fully sequenced AAV2 and partly sequenced AAV13. When AAV integration occurs, only a fragment of the AAV genome is detectable in later DNA or RNA sequencing. The integrated fragment is typically from the 3’ end of the AAV genome, and this positional bias has been only partly explained. Three research groups searched for evidence of AAV integration in HCC RNAseq samples in the Cancer Genome Atlas (TCGA) but reported conflicting results.ResultsWe collected and analyzed whole transcriptome and viral capture DNA sequencing in paired tumor and non-tumor samples from two liver cancer Asian cohorts from Thailand (N = 147, 47 HCC and 100 intrahepatic cholangiocarcinoma (iCCA)) and Mongolia (N = 70, all HCC). We found only one HCC patient with a potentially oncogenic integration of AAV, in contrast to higher frequency reported in European patients. There were no oncogenic AAV integrations in iCCA patients. AAV genomic segments are present preferentially in the non-tumor samples of Thai patients.By analyzing the AAV genome positions of oncogenic and non-oncogenic integrated fragments, we found that almost all the putative oncogenic integrations overlap the X gene, which is present and functional only in the strain AAV2 among all fully sequenced strains. This gene content difference could explain why putative oncogenic integrations from other AAV strains have not been reported.We resolved the discrepancies in previous analyses of AAV presence in TCGA HCC samples and extended it to CCA. There are 12 TCGA samples with an AAV segment and none are in Asian patients. AAV segments are present in preferentially in TCGA non-tumor samples, like what we observed in the Thai patients.ConclusionsOur findings suggest a minimal AAV risk of hepatocarcinogenesis in Asian liver cancer patients. The partial genome presence and positional bias of AAV integrations into the human genome has complicated analysis of possible roles of AAV in liver cancer.

Abstract 2658: Oncogenicity of adeno-associated virus (AAV) integration into the genomes of liver cancer patients depends on the overlap between the AAV integration site and the AAV X gene

Abstract Viruses may affect human health by altering host immunity and the genome, contributing to chronic disease and cancer. The purpose of this study was to examine evidence of oncogenicity accompanying integrations of non-hepatitis viruses into the liver cancer genome through analysis of a cohort of 147 Thai liver cancer patients comprised of 47 hepatocellular carcinoma (HCC) cases and 100 intrahepatic cholangiocarcinoma (iCCA) cases. For each case, we generated paired RNASeq data from cancerous tissue and adjacent non-cancerous tissue. Initial analysis with PathSeq comparing viruses with RefSeq genomes revealed that only Adeno-associated viruses (AAV) were differentially and predominantly present in the non-cancer samples (p < 9.4e-6, two-sided Fisher test). However, J. Zucman-Rossi and colleagues (Nault et al., Nat. Genet. 2015; 47:1187-1193) reported that integrations of AAV resembling the strains AAV2 or AAV13 near human genes such as CCNA2, CCNE1 are oncogenic. Understanding when AAV integration is oncogenic is important because AAV2-derived vectors are used in gene therapy. To investigate the apparent discrepancy, we performed AAV-targeted viral capture DNA sequencing of the cancerous tissue and adjacent non-cancerous tissue from each patient with AAV. One patient has an integration of AAV2/13 into CCNA2, expanded in the non-cancer sample and further expanded in the cancer sample that has 10,006 chimeric AAV/CCNA2 DNA reads. We validated that this integration substantially increased mRNA expression of CCNA2, replicating the previous findings. In contrast, we found ten patients with AAV integrations into other loci only in the non-cancer samples and two other patients with AAV integrations into other loci only in the cancer samples. These single-sample AAV integrations did not significantly increase the expression of the target gene, if any, suggesting that they are not oncogenic. All previously reported AAV integrations have been demonstrated only in HCC. Similarly, in our analysis, the only oncogenic integration was in a patient with HCC. Among the ten non-oncogenic integrations in non-cancer samples, one is HCC and nine are iCCA. Both non-oncogenic integrations in cancer samples were in iCCA. Analysis of all previously published AAV integrations and the new AAV integrations we identified showed that almost all the integrations reported as oncogenic overlap the poorly characterized X gene, while the non-oncogenic integrations need not overlap the X gene. This finding explains why the oncogenic AAV integrations are necessarily from AAV2 or a similar strain since other sequenced AAV strains do not contain the X gene. This would suggest re-engineering AAV vectors to make the X gene non-functional, to retain the useful properties of these vectors and concomitantly mitigate the risk of liver cancer in AAV-therapy recipients. Citation Format: Alejandro A. Schaffer, Dana Dominguez, E. M. Gertz, Lesley M. Chapman, Anuradha Budhu, Marshonna Forgues, Jittiporn Chaisaingmongkol, Siritida Rabibhadana, Benjarath Pupacdi, Xiaolin Wu, Curtis C. Harris, Mathuros Ruchirawat, Eytan Ruppin, Xin W. Wang. Oncogenicity of adeno-associated virus (AAV) integration into the genomes of liver cancer patients depends on the overlap between the AAV integration site and the AAV X gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2658.

AAV8 transduction capacity is reduced by prior exposure to endosome-like pH conditions.

Adeno-associated virus (AAV) is an essential instrument in the neuroscientist's toolkit, which allows delivery of DNA to provide labeling with fluorescent proteins or genetic instructions to regulate gene expression. In the field of neural regeneration, the transduction of neurons enables the observation and regulation of axon growth and regeneration, and in the future will likely be a mechanism for delivering molecular therapies to promote sprouting and regeneration after central nervous system injury. Traditional formulations of AAV preparations permit efficient viral transduction under physiologic conditions, but an improved understanding of the mechanistic limitations of AAV transduction may facilitate production of more resilient AAV strains for investigative and therapeutic purposes. We studied AAV transduction in the context of prior exposure of AAV serotype 8 (AAV8) to environmental pH within the range encountered during endosomal endocytosis (pH 7.4 to pH 4.4), during which low pH-triggered structural and autoproteolytic changes to the viral capsid are believed to be necessary for endosome escape and virus uncoating. Due to the fundamental nature of these processes, we hypothesized that premature exposure of AAV8 particles to acidic pH would decrease viral transduction of HT1080 cells in vitro, as measured by fluorescent reporter gene expression using high-content imaging analysis. We found that increasingly acidic incubation conditions were associated with concomitant reductions in transduction efficiency, and that quantitative levels of reporter gene expression in transduced cells were similarly decreased. The biggest decrease in transduction occurred between pH 7.4 and pH 6.4, suggesting the possible co-occurrence of a pH-associated event and viral inactivation within that range. Taken together, these findings indicate that exposure of AAV8 to acidic pH for as little as 1 hour is deleterious to transduction ability. Future studies are necessary to understand the pH-associated causative mechanisms involved. This study was approved by the University of Miami Institutional Animal Care and Use Committee, USA (Protocol #18-108-LF) on July 12, 2018.

Systemic and Persistent Muscle Gene Expression in Rhesus Monkeys with a Liver De-Targeted Adeno-Associated Virus Vector.

The liver is a major off-target organ in gene therapy approaches for cardiac and musculoskeletal disorders. Intravenous administration of most of the naturally occurring adeno-associated virus (AAV) strains invariably results in vector genome sequestration within the liver. In the current study, we compared the muscle tropism and transduction efficiency of a liver de-targeted AAV variant to AAV9 following systemic administration in newborn rhesus monkeys. In vivo bioluminescence imaging was performed to monitor transgene expression (firefly luciferase) post administration. Results indicated comparable and sustained levels of systemic firefly luciferase gene expression in skeletal muscle over a period of two years. Quantitation of vector biodistribution in harvested tissues post-administration revealed widespread recovery of vector genomes delivered by AAV9 but markedly decreased levels in major systemic organs from the AAV variant. These studies validate the translational potential and safety of liver de-targeted AAV strains for gene therapy of muscle-related diseases.

96. An Approach to Compare Multiple Adeno-Associated Virus (AAV) Capsids Side-by-Side in a Single Eye Following Intravitreal Injection

Current gene therapy approaches for ocular diseases rely largely on the use of subretinal injection for treatment of outer nuclear layer cells of interest. While subretinal injection allows for efficient transduction of retinal cells close to the site of the injection, there is a significant risk that the injection can further damage retinas with compromised integrity. Several studies have identified new adeno-associated virus (AAV) capsid mutants that are potentially able to transduce outer nuclear layer cells following intravitreal injections. To further explore AAV vector-mediated intravitreal injection approaches for clinical translation, it is imperative to directly compare retinal transduction efficiencies between newly identified and previously published AAV mutants. Using clinically relevant large animal models, these mutant capsids, as well as other commonly used AAV serotypes, can be tested in a comprehensive manner. To this end, we propose to utilize a viral DNA/RNA barcoding approach established by our lab. In this approach, AAV capsids derived from different serotypes and mutants can be identified by DNA and RNA barcodes unique to each AAV capsid. These barcodes are contained in viral genomes and also expressed as RNA barcodes following cellular transduction. As each AAV capsid strain has a unique viral genome, a mass transduction of a number of different AAV strains can be conducted in order to directly compare transduction levels side-by-side in a single eye. To test feasibility of the barcode approach and establish proof-of-principle in the ocular gene therapy research, we produced a DNA/RNA-barcoded AAV library that contained 20 serotypes including AAV serotypes 1-11 and AAV mutants including DJ, LK03, 2i8 and 2G9. Each AAV strain expresses a pair of 12 nucleotide-long RNA barcodes unique to each strain under the control of the human U6 snRNA promoter. Seven C57BL/6J mice have been injected into vitreous humor bilaterally with the DNA/RNA-barcoded AAV library vector at doses ranging from 2 to 9 x109 vg. To date, eyes were harvested from one mouse 3 weeks post injection. Total DNA and RNA were extracted from the retina as a whole from each eye, and were subjected to DNA-PCR or reverse transcriptase (RT)-PCR to amplify viral DNA and RNA barcodes, followed by Illumina barcode sequencing. A preliminary data analysis revealed that AAV2, AAV-DJ and AAV2G9 transduced the murine retina better than other AAV strains including AAV9, the reference control AAV strain contained in the library. Although further optimization of the approach may be required to move this project forward, we plan to apply this method to assess transduction efficiencies of various AAV strains side by side in specific subsets of sorted retinal cells and in various animal species. The safer approach of intravitreal injections with the most optimal AAV vectors would significantly increase the ability to treat over 200 genetic retinal diseases and therefore the data that will be obtained from these studies will considerably benefit the field of retinal gene therapy.

255. A Method for Developing More Potent AAV Capsids by Prolonging the Vector Half-Lives in the Blood Circulation

Adeno-associated virus (AAV) has been used successfully as a vector for in vivo gene therapy. Many different serotypes are under investigation, but AAV9 vectors especially show robust in vivo transduction by systemic administration via the periphery. Although the mechanism of this robustness is not yet completely understood, the superior ability to cross the endothelial cell barriers including the blood-brain barrier has been presumed to play a key role in efficient systemic gene delivery by AAV9. In this regard, our laboratory has accumulated provocative evidence that AAV permeability across the endothelium is not substantially different among many AAV strains (serotypes and mutants) per se, but that AAV9's in vivo robustness following intravascular administration is due to its distinctively delayed blood clearance. Given that many AAV strains transduce cells better than AAV9 when directly injected into the parenchyma, it should be reasonable to hypothesize that prolonging the half-life of an AAV could substantially increase in vivo transduction efficiency following systemic administration. Here, we show that substitution of a small subset of amino acids in the C-terminal half of the viral capsid can substantially prolong the half-life of AAV1 in the blood and confer a superior in vivo transduction efficiency that is better than that of AAV9 following systemic vector administration. Based on the results from our preliminary AAV1 and AAV9 capsid domain swapping study and a capsid amino acid sequence alignment study, we identified 7 capsid segments comprising 5 to 23 amino acids that might contribute to the persistent circulation of AAV9 in the blood. Each of these 7 segments can be replicated in the AAV1 capsid, which shows a rapid blood clearance rate, by substituting 3 to 11 amino acids of the AAV1 capsid with those of the AAV9 capsid. In the study, we created a panel of 27=128 AAV1-derived strains that carry these segment(s) in any possible combinations (note: one of the 128 is the same as the wild-type AAV1 capsid). We then determined blood clearance rates of all the 128 AAV1-derived strains and AAV9 following intravenous injection into mice using the AAV Barcode-Seq approach. The results show that 42 mutants that showed a >30-fold increase in persistence in the blood at 24 hours post-injection compared to the parental wild-type AAV1 all shared either of the following segment combinations, xxx9xx9 or xxxxx99 (note: each letter represents a segment and × can be either AAV1 or AAV9). This indicates that the 7th segment plays an indispensable role but requires either of the 4th and 6th segments for substantially prolonging the half-life. A preliminary tissue transduction analysis in mice revealed that these AAV1-derived mutants with a long half-life showed enhanced in vivo gene delivery compared to AAV9. In a separate study, we also identified a 16 amino acid-long segment in the N-terminal half of the AAV9 capsids that is essential for maintaining high levels of circulating viral particles in the blood at 72 hours post-injection. We are currently investigating whether this amino acid motif, when combined with the above-described xxx9xx9 or xxxxx99 motifs, could further enhance persistence of AAV1-derived mutants and augment in vivo transduction, using another set of 24 AAV1-derived composite mutant capsids in mice. In summary, the study establishes proof-of-principle of a new approach to create more portent AAV vectors by modulating the vector half-life. This approach will be particularly useful for developing novel AAV capsids that can transduce the brain tissues more efficiently by systemic vector administration.

An emerging adeno-associated viral vector pipeline for cardiac gene therapy.

The naturally occurring adeno-associated virus (AAV) isolates display diverse tissue tropisms in different hosts. Robust cardiac transduction in particular has been reported for certain AAV strains. Successful applications of these AAV strains in preclinical and clinical settings with a focus on treating cardiovascular disease continue to be reported. At the same time, these studies have highlighted challenges such as cross-species variability in AAV tropism, transduction efficiency, and immunity. Continued progress in our understanding of AAV capsid structure and biology has provided the rationale for designing improved vectors that can possibly address these concerns. The current report provides an overview of cardiotropic AAV, existing gaps in our knowledge, and newly engineered AAV strains that are viable candidates for the cardiac gene therapy clinic.

Engraftment of a Galactose Receptor Footprint onto Adeno-associated Viral Capsids Improves Transduction Efficiency

New viral strains can be evolved to recognize different host glycans through mutagenesis and experimental adaptation. However, such mutants generally harbor amino acid changes that affect viral binding to a single class of carbohydrate receptors. We describe the rational design and synthesis of novel, chimeric adeno-associated virus (AAV) strains that exploit an orthogonal glycan receptor for transduction. A dual glycan-binding AAV strain was first engineered as proof of concept by grafting a galactose (Gal)-binding footprint from AAV serotype 9 onto the heparan sulfate-binding AAV serotype 2. The resulting chimera, AAV2G9, continues to bind heparin affinity columns but interchangeably exploits Gal and heparan sulfate receptors for infection, as evidenced by competitive inhibition assays with lectins, glycans, and parental AAV strains. Although remaining hepatotropic like AAV2, the AAV2G9 chimera mediates rapid onset and higher transgene expression in mice. Similarly, engraftment of the Gal footprint onto the laboratory-derived strain AAV2i8 yielded an enhanced AAV2i8G9 chimera. This new strain remains liver-detargeted like AAV2i8 while selectively transducing muscle tissues at high efficiency, comparable with AAV9. The AAV2i8G9 chimera is a promising vector candidate for targeted gene therapy of cardiac and musculoskeletal diseases. In addition to demonstrating the modularity of glycan receptor footprints on viral capsids, our approach provides design strategies to expand the AAV vector toolkit.

The AAV vector toolkit: poised at the clinical crossroads.

The discovery of naturally occurring adeno-associated virus (AAV) isolates in different animal species and the generation of engineered AAV strains using molecular genetics tools have yielded a versatile AAV vector toolkit. Promising results in preclinical animal models of human disease spurred the much awaited transition toward clinical application, and early successes in phase I/II clinical trials for a broad spectrum of genetic diseases have recently been reported. As the gene therapy community forges ahead with cautious optimism, both preclinical and clinical studies using first generation AAV vectors have highlighted potential challenges. These include cross-species variation in vector tissue tropism and gene transfer efficiency, pre-existing humoral immunity to AAV capsids and vector dose-dependent toxicity in patients. A battery of second generation AAV vectors, engineered through rational and combinatorial approaches to address the aforementioned concerns, are now available. This review will provide an overview of preclinical studies with the ever-expanding AAV vector portfolio in large animal models and an update on new lead AAV vector candidates poised for clinical translation.

Transduction, Tropism, and Biodistribution of AAV Vectors in the Lacrimal Gland

The lacrimal gland (LG) delivers defensive and metabolic factors to the ocular surface. These functions may be disrupted in several diseases, and for most of them there is no cure. The aim of this study is to investigate conditions and limitations for using adeno-associated virus (AAV) vectors as gene transfer agents to LG. Eight-week-old Balb/c mice were used to investigate route, gene expression, and time course of AAV gene vector transfer to LG. AAV vectors encoding firefly luciferase were administered to the LG and luciferase expression was evaluated in vivo by immunohistochemistry. Ocular surface and neutralizing antibodies were also evaluated. The present work revealed that AAV vectors are able to delivery DNA to the LGs of mice. Direct injection had the highest level of transduction, and topical ocular drops the lowest. Overall, the AAV strain with highest transduction activity as measured by both luminescence and immunohistochemistry was AAV9, followed by AAV 5w8 and AAV5. Transduction was not different between sexes, could be detected as soon as 24 hours after injection, and lasted for at least 30 days (study termination). No tissue damage was observed when compared with controls. All vectors with detectable LG transduction induced neutralizing antibodies. LG gene delivery by AAV vectors appears to be both safe and well tolerated. The choice of vector influences both the overall transduction activity, as well as the spread of vector to other organs. This work supports the use of AAV-mediated gene therapy for dry eye.

Reengineering a receptor footprint of adeno-associated virus enables selective and systemic gene transfer to muscle

Reengineering the receptor footprints of adeno-associated virus (AAV) isolates may yield variants with improved properties for clinical applications. We generated a panel of synthetic AAV2 vectors by replacing a hexapeptide sequence in a previously identified heparan sulfate receptor footprint with corresponding residues from other AAV strains. This approach yielded several chimeric capsids displaying systemic tropism after intravenous administration in mice. Of particular interest, an AAV2/AAV8 chimera designated AAV2i8 displayed an altered antigenic profile, readily traversed the blood vasculature, and selectively transduced cardiac and whole-body skeletal muscle tissues with high efficiency. Unlike other AAV serotypes, which are preferentially sequestered in the liver, AAV2i8 showed markedly reduced hepatic tropism. These features of AAV2i8 suggest that it is well suited to translational studies in gene therapy of musculoskeletal disorders.