Paul E. Monahan

Adeno-associated virus vectors for gene therapy: more pros than cons?

Gene therapy vectors based on the adeno-associated virus (AAV) are being developed for a widening variety of therapeutic applications. Enthusiasm for AAV is due, not only to the relative safety of these vectors, but also to advances in understanding of the unique biology of this virus. This review examines a number of long-standing concerns regarding the utility of AAV for gene transfer in light of many new insights into the biology, immunology and production of AAV.

Direct intramuscular injection with recombinant AAV vectors results in sustained expression in a dog model of hemophilia

A recombinant adeno-associated virus (rAAV) vector carrying the human factor IX cDNA was tested for safety and therapeutic gene expression in a canine model of human hemophilia B. Intramuscular delivery of rAAV was chosen based on our previous work which described long-term (>1.5 years) reporter gene expression in immunocompetent mice following direct muscle injection. For the current study, rAAV with the human factor IX (hF.IX) cDNA under the control of the cytomegalovirus (CMV) immediate–early promoter was constructed, and rAAV/hF.IX proved capable of transducing hemophilic dog primary fibroblast cultures in a dose-dependent fashion. Direct intramuscular injection of 2.5 × 1012 rAAV/hF.IX virus particles into the hindlimbs of a hemophilia B dog was tolerated without bleeding or systemic reaction, and the animal was asymptomatic throughout the entire study. Transient reduction in the whole blood clotting time (WBCT) occurred during the first week, with the anticipated development of an antihuman F.IX inhibitor antibody which corresponded with the loss of coagulation correction. At 10 weeks after vector administration, immunohistochemical analysis of injected muscle confirmed continued hF.IX expression. Limited areas of focal lymphocytic infiltration and myofiber pathology were detected which directly correlated with positive antibody staining for helper adenovirus contamination. PCR tissue analysis revealed rAAV/hF.IX DNA solely in injected muscle tissue and adjacent lymph node, without dissemination to other organs (including gonads). This first large animal study suggests that intramuscular gene delivery using rAAV vectors is safe and supports continued development of this approach for gene therapy of human diseases, including hemophilia B.

Phase 1 Gene Therapy for Duchenne Muscular Dystrophy Using a Translational Optimized AAV Vector

Efficient and widespread gene transfer is required for successful treatment of Duchenne muscular dystrophy (DMD). Here, we performed the first clinical trial using a chimeric adeno-associated virus (AAV) capsid variant (designated AAV2.5) derived from a rational design strategy. AAV2.5 was generated from the AAV2 capsid with five mutations from AAV1. The novel chimeric vector combines the improved muscle transduction capacity of AAV1 with reduced antigenic crossreactivity against both parental serotypes, while keeping the AAV2 receptor binding. In a randomized double-blind placebo-controlled phase I clinical study in DMD boys, AAV2.5 vector was injected into the bicep muscle in one arm, with saline control in the contralateral arm. A subset of patients received AAV empty capsid instead of saline in an effort to distinguish an immune response to vector versus minidystrophin transgene. Recombinant AAV genomes were detected in all patients with up to 2.56 vector copies per diploid genome. There was no cellular immune response to AAV2.5 capsid. This trial established that rationally designed AAV2.5 vector was safe and well tolerated, lays the foundation of customizing AAV vectors that best suit the clinical objective (e.g., limb infusion gene delivery) and should usher in the next generation of viral delivery systems for human gene transfer.

Persistent expression of canine factor IX in hemophilia B canines.

We previously demonstrated that direct intramuscular injection of recombinant adeno-associated virus (rAAV) carrying the human FIX (hFIX) cDNA can safely be administered to hemophilic B canines and express human factor IX protein; however, the functional activity of the hFIX protein could not be assessed due to anti-human FIX antibody (inhibitor) formation. To test the therapeutic efficacy of rAAV in hemophilic dogs, rAAV type 2 (rAAV2) carrying canine FIX (cFIX) cDNA was injected into the skeletal muscle of two dogs at doses of 1012–13particles. Circulating cFIX protein levels were maintained for 1 year at levels of 1–2% of normal. Hemostatic correction (WBCT and APTT) paralleled plasma FIX antigen levels. Both dogs still required plasma infusion for spontaneous and traumatic bleeding events. Inhibitors to cFIX protein were not detected in either animal by Bethesda assay. Neutralizing antibodies directed against AAV-2 capsid were pronounced and persistent. Vector DNA and mRNA transcripts were detected only at the injected skeletal muscle tissue. Analysis of both high and low molecular weight DNA identified both replicative episomal and integrated AAV species. These results demonstrate that persistent secretion of the FIX transgene protein, necessary for successful gene therapy of hemophilia B, can be achieved using the parvovirus-based rAAV vector

Neutralizing antibodies against adeno-associated virus examined prospectively in pediatric patients with hemophilia

Recombinant adeno-associated virus (rAAV) is a promising gene delivery vector and has recently been used in patients with hemophilia. One limitation of AAV application is that most humans have experienced wild-type AAV serotype 2 exposure, which frequently generates neutralizing antibodies (NAbs) that may inhibit rAAV2 vector transduction. Employing alternative serotypes of rAAV vectors may circumvent this problem. We investigated the development of NAbs in early childhood by examining sera gathered prospectively from 62 children with hemophilia A, participating in a multi-institutional hemophilia clinical trial (the Joint Outcome Study). Clinical applications in hemophilia therapy have been suggested for serotypes AAV2, AAV5 and AAV8, therefore NAbs against these serotypes were serially assayed over a median follow-up of 4 years. NAbs prevalence increased during early childhood for all serotypes. NAbs against AAV2 (43.5%) were observed more frequently and at higher titers compared with both AAV5 (25.8%) and AAV8 (22.6%). NAbs against AAV5 or AAV8 were rarely observed in the absence of co-prevalent and higher titer AAV2 NAbs, suggesting that NAbs to AAV5 and AAV8 were detected following AAV2 exposure due to partial cross-reactivity of AAV2-directed NAbs. The results may guide rational design of clinical trials using alternative AAV serotypes and suggest that younger patients who are given AAV gene therapy will benefit from the lower prevalence of NAbs.

Proteasome Inhibitors Enhance Gene Delivery by AAV Virus Vectors Expressing Large Genomes in Hemophilia Mouse and Dog Models: A Strategy for Broad Clinical Application

Delivery of genes that are larger than the wild-type adeno-associated virus (AAV) 4,681 nucleotide genome is inefficient using AAV vectors. We previously demonstrated in vitro that concurrent proteasome inhibitor (PI) treatment improves transduction by AAV vectors encoding oversized transgenes. In this study, an AAV vector with a 5.6 kilobase (kb) factor VIII expression cassette was used to test the effect of an US Food and Drug Administration-approved PI (bortezomib) treatment concurrent with vector delivery in vivo. Intrahepatic vector delivery resulted in factor VIII expression that persisted for >1 year in hemophilia mice. Single-dose bortezomib given with AAV2 or AAV8 factor VIII vector enhanced expression on average ~600 and ~300%, respectively. Moreover, coadministration of AAV8.canineFVIII (1 × 10(13) vg/kg) and bortezomib in hemophilia A dogs (n = 4) resulted in normalization of the whole blood clotting time (WBCT) and 90% reduction in hemorrhages for >32 months compared to untreated hemophilia A dogs (n = 3) or dogs administered vector alone (n = 3). Demonstration of long-term phenotypic correction of hemophilia A dogs with combination adjuvant bortezomib and AAV vector expressing the oversized transgene establishes preclinical studies that support testing in humans and provides a working paradigm to facilitate a significant expansion of therapeutic targets for human gene therapy.

A systematic approach to controlling problem bleeds in patients with severe congenital haemophilia A and high-titre inhibitors

Summary.  The presence of inhibitory antibodies to clotting factors complicates the treatment of bleeding in haemophilia patients. For patients with high‐titre inhibitors, bypassing agents are essential to haemostatic management. To determine optimal treatment practices, an international panel of physicians convened to develop a systematic treatment approach for problem bleeds (i.e. bleeds that are unresponsive to initial therapy with a single agent within a reasonable amount of time) in haemophilia patients with inhibitors. Aim: The goal of this panel was to develop a consensus algorithm that would aid physicians in considering a variety of treatment approaches to optimize patient care by preventing extensive therapy with inadequate treatments that may lead to suboptimal patient outcomes and unnecessary costs. Methods: Consensus opinions were analyzed for clinical preferences at different time periods, depending on patient response to treatment. Decision‐making points were defined based on the type of bleed: every 8–12 h for the first 24 h, then every 24 h thereafter for limb‐threatening bleeds; every 2–4 h for 2–7 days for life‐threatening bleeds. Results: The resultant consensus guidelines provide a generalized methodology to guide the treatment of problem bleeds in patients with severe haemophilia A and inhibitors, and emphasize changing treatment at the first sign of an inadequate haemostatic response. The treatment algorithms apply to both paediatric and adult patients, although the differences between the two groups were reviewed. Conclusion: These guidelines are focused on optimising the timing of treatment decisions, which may lead to faster responses and improved outcomes.

Epigenetic activation of unintegrated HIV-1 genomes by gut-associated short chain fatty acids and its implications for HIV infection

Integration of HIV-1 linear DNA into the host chromatin is an essential step in the viral life cycle. However, the majority of reverse-transcribed, nuclear-imported viral genomes remain episomal, either as linear or circular DNA. To date, these nonintegrated viral genomes are largely considered “dead-end products” of reverse transcription. Indeed, limited gene expression from nonintegrated HIV-1 has been reported, although the mechanism that renders nonintegrating HIV-1 genomes incapable of supporting efficient viral replication has not been fully elucidated. Here, we demonstrate that nonintegrating HIV-1 and HIV-1-based vector genomes are organized into chromatin structures and enriched with histone modifications typical of transcriptionally silenced chromatin. Gene expression and replication of nonintegrating HIV-1 was notably increased in vitro upon exposure to histone deacetylase inhibitors (HDACi) in the form of various short-chain fatty acids (SCFAs) known to be endogenously produced by normal microbial-gut flora. Furthermore, we demonstrated genetic and functional crosstalk between episomal and integrated vector/viral genomes, resulting in recombination between integrated and nonintegrated HIV-1, as well as mobilization of episomal vector genomes by productive viral particles encoded by integrated viral genomes. Finally, we propose a mechanism describing the role of episomal HIV-1 forms in the viral life cycle in a SCFA-rich gut environment.

Cellular immune response to cryptic epitopes during therapeutic gene transfer

The immune response has been implicated as a critical factor in determining the success or failure of clinical gene therapy trials. Generally, such a response is elicited by the desired transgene product or, in some cases, the delivery system. In the current study, we report the previously uncharacterized finding that a therapeutic cassette currently being used for human investigation displays alternative reading frames (ARFs) that generate unwanted protein products to induce a cytotoxic T lymphocyte (CTL) response. In particular, we tested the hypothesis that antigenic epitopes derived from an ARF in coagulation factor IX (F9) cDNA can induce CTL reactivity, subsequently killing F9-expressing hepatocytes. One peptide (p18) of 3 candidates from an ARF of the F9 transgene induced CD8+ T cell reactivity in mice expressing the human MHC class I molecule B0702. Subsequently, upon systemic administration of adeno-associated virus (AAV) serotype 2 vectors packaged with the F9 transgene (AAV2/F9), a robust CD8+ CTL response was elicited against peptide p18. Of particular importance is that the ARF epitope-specific CTLs eliminated AAV2/F9-transduced hepatocytes but not AAV2/F9 codon-optimized (AAV2/F9-opt)-transduced liver cells in which p18 epitope was deleted. These results demonstrate a previously undiscovered mechanism by which CTL responses can be elicited by cryptic epitopes generated from a therapeutic transgene and have significant implications for all gene therapy modalities. Such unforeseen epitope generation warrants careful analysis of transgene sequences for ARFs to reduce the potential for adverse events arising from immune responses during clinical gene therapy protocols.

Safety of adeno-associated virus gene therapy vectors: a current evaluation.

An increasing number of strategies for molecular treatment of disease rely on the adeno-associated virus (AAV) as a therapeutic gene delivery vector. One of the most attractive features of this viral DNA vector is the perceived safety of AAV gene delivery. Recent applications in human clinical trials support the safety record established in preclinical trials, with evidence of gene transfer in the absence of cellular immune responses or tissue disturbance. Nevertheless, many aspects of the biology of the wild type AAV and its derivatives are still being explored. While the therapeutic potential of novel recombinant AAV therapeutics appears promising, recent insights suggest aspects of their pharmacokinetics, biodistribution and toxicity that require consideration to achieve the safest application of these molecular medicines.