Rita Sarkar

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.

Gene transfer to hemophilia A mice via oral delivery of FVIII–chitosan nanoparticles

Effective oral delivery of a non-viral gene carrier would represent a novel and attractive strategy for therapeutic gene transfer. To evaluate the potential of this approach, we studied the oral gene delivery efficacy of DNA polyplexes composed of chitosan and Factor VIII DNA. Transgene DNA was detected in both local and systemic tissues following oral administration of the chitosan nanoparticles to hemophilia A mice. Functional factor VIII protein was detected in plasma by chromogenic and thrombin generation assays, reaching a peak level of 2-4% FVIII at day 22 after delivery. In addition, a bleeding challenge one month after DNA administration resulted in phenotypic correction in 13/20 mice given 250-600 microg of FVIII DNA in chitosan nanoparticles, compared to 1/13 mice given naked FVIII DNA and 0/6 untreated mice. While further optimization would be required to render this type of delivery system practical for hemophilia A gene therapy, the findings suggest the feasibility of oral, non-viral delivery for gene medicine applications.

Efficacy and Safety of Long-term Prophylaxis in Severe Hemophilia A Dogs Following Liver Gene Therapy Using AAV Vectors

Developing adeno-associated viral (AAV)-mediated gene therapy for hemophilia A (HA) has been challenging due to the large size of the factor VIII (FVIII) complementary DNA and the concern for the development of inhibitory antibodies to FVIII in HA patients. Here, we perform a systematic study in HA dogs by delivering a canine FVIII (cFVIII) transgene either as a single chain or two chains in an AAV vector. An optimized cFVIII single chain delivered using AAV serotype 8 (AAV8) by peripheral vein injection resulted in a dose-response with sustained expression of FVIII up to 7% (n = 4). Five HA dogs administered two-chain delivery using either AAV8 or AAV9 via the portal vein expressed long-term, vector dose-dependent levels of FVIII activity (up to 10%). In the two-chain approach, circulating cFVIII antigen levels were more than fivefold higher than activity. Notably, no long-term immune response to FVIII was observed in any of the dogs (1/9 dogs had a transient inhibitor). Long-term follow-up of the dogs showed a remarkable reduction (>90%) of bleeding episodes in a combined total of 24 years of observation. These data demonstrate that both approaches are safe and achieve dose-dependent therapeutic levels of FVIII expression, which supports translational studies of AAV-mediated delivery for HA.

Absence of a desmopressin response after therapeutic expression of factor VIII in hemophilia A dogs with liver-directed neonatal gene therapy

Hemophilia A (HA) is a bleeding disorder caused by factor VIII (FVIII) deficiency. FVIII replacement therapy can reduce bleeding but is expensive, inconvenient, and complicated by development of antibodies that inhibit FVIII activity in 30% of patients. Neonatal hepatic gene therapy could result in continuous secretion of FVIII into blood and might reduce immunological responses. Newborn HA mice and dogs that were injected i.v. with a retroviral vector (RV) expressing canine B domain-deleted FVIII (cFVIII) achieved plasma cFVIII activity that was 139 ± 22% and 116 ± 5% of values found in normal dogs, respectively, which was stable for 1.5 yr. Coagulation tests were normalized, no bleeding had occurred, and no inhibitors were detected. This is a demonstration of long-term fully therapeutic gene therapy for HA in a large animal model. Desmopressin (DDAVP; 1-deamino-[d-Arg8]vasopressin) is a drug that increases FVIII activity by inducing release of FVIII complexed with von Willebrand factor from endothelial cells. It has been unclear, however, if the FVIII is synthesized by endothelial cells or is taken up from blood. Because the plasma cFVIII in these RV-treated dogs derives primarily from transduced hepatocytes, they provided a unique opportunity to study the biology of the DDAVP response. Here we show that DDAVP did not increase plasma cFVIII levels in the RV-treated dogs, although von Willebrand factor was increased appropriately. This result suggests that the increase in FVIII in normal dogs after DDAVP is due to release of FVIII synthesized by endothelial cells.

Transplantation of endothelial cells corrects the phenotype in hemophilia A mice

Summary.  Background: The deficiency of factor VIII, a co‐factor in the intrinsic coagulation pathway results in hemophilia A. Although FVIII is synthesized largely in the liver, the specific liver cell type(s) responsible for FVIII production is controversial. Objective: This study aimed to determine the cellular origin of FVIII synthesis and release in mouse models. Methods: We transplanted cells into the peritoneal cavity of hemophilia A knockout mice. Plasma FVIII activity was measured using a Chromogenix assay 2–7 days after cell transplantation, and phenotypic correction was determined with tail‐clip challenge 7 days following cell transplantation. Transplanted cells were identified by histologic and molecular assays. Results: Untreated hemophilia A mice, as well as mice treated with the hepatocyte‐enriched fraction, showed extensive mortality following tail‐clip challenge. In contrast, recipients of unfractionated liver cells (mixture of hepatocytes, liver sinusoidal endothelial cells (LSEC), Kupffer cells, and hepatic stellate cells) or of the cell fraction enriched in LSECs survived tail‐clip challenge (P < 0.001). FVIII was secreted in the blood stream in recipients of unfractionated liver cells, LSECs and pancreatic islet‐derived MILE SVEN 1 (MS1) endothelial cells. Although transplanted hepatocytes maintained functional integrity in the peritoneal cavity, these cells did not produce detectable plasma FVIII activity. Conclusions: The assay of cell transplantation in the peritoneal cavity showed that endothelial cells but not hepatocytes produced phenotypic correction in hemophilia A mice. Therefore, endothelial cells should be suitable additional targets for cell and gene therapy in hemophilia A.

Partial Correction of Murine Hemophilia A with Neo-Antigenic Murine Factor VIII

We have previously reported a factor VIII knockout (FVIII KO) mouse model for hemophilia A. Here we demonstrate the presence of nonfunctional heavy chain factor VIII protein in the mouse, making it an excellent model for cross-reacting material (CRM)-positive hemophilia A patients, who express normal levels of a dysfunctional FVIII protein. We attempted to correct these mice phenotypically by transduction of wild-type mouse factor VIII cDNA delivered in an E1/E3-deleted adenoviral vector by tail vein injection. All treated mice displayed initial high-level FVIII expression that diminished after 1 month. Ten of 12 mice administered between 6 x 10(9) and 1 x 10(11) particles/mouse along with anti-CD4 antibody showed long-term FVIII activity (0.03-0.05 IU/ml, equivalent to 3-5% of normal FVIII) that corrected the phenotype. Wild-type murine FVIII was a neo-antigen to the KO mice, generating both cytotoxic and humoral immune responses. Immune suppression with anti-CD4 antibody abrogated these immune responses. These data demonstrate that despite the presence of endogenous FVIII protein the immune system still recognizes a species-specific transgene protein as a neo-antigen, eliciting a cytotoxic T cell response. This phenomenon may exist in the treatment of other genetic disorders by gene therapy.

Complete Correction of Hemophilia A with Adeno-Associated Viral Vectors Containing a Full-Size Expression Cassette

Hemophilia A is caused by a deficiency in the factor VIII (FVIII) gene. Constrained by limited packaging capacity, even the 4.3-kb B domain-deleted FVIII remained a challenge for delivery by a single adeno-associated viral (AAV) vector. Studies have shown that up to a 6.6-kb vector sequence may be packaged into AAV virions, which suggested an alternative strategy for hemophilia A gene therapy. To explore the usefulness of AAV vectors carrying an oversized FVIII gene, we constructed the AAV-FVIII vector under the control of a beta-actin promoter with a cytomegalovirus enhancer (CB) and a bovine growth hormone (bGH) poly(A) sequence. The CB promoter plus bGH signal was shown to be 3- to 5-fold more potent than the mini-transthyretin (TTR) promoter with a synthetic poly(A) sequence for directing FVIII expression in the liver. Despite the 5.75-kb genome size of pAAV-CB-FVIII, sufficient AAV vectors were produced for in vivo testing. Approximately 3- to 5-fold more FVIII secretion was observed in animals receiving AAV-CB-FVIII vectors than in those receiving standard-sized AAV-TTR-FVIII vectors. Both the activated partial thromboplastin time assay and the whole blood thromboelastographic analysis confirmed that AAV-FVIII vectors fully corrected the bleeding phenotype of hemophilia mice. These results suggest that AAV vectors with an oversized genome should be useful for not only hemophilia A gene therapy but also other diseases with large cDNA such as muscular dystrophy and cystic fibrosis.

A single adeno-associated virus (AAV)-murine factor VIII vector partially corrects the hemophilia A phenotype

Summary.  A major obstacle for delivery of factor (F)VIII using adeno‐associated virus (AAV) vectors is the large size of FVIII cDNA, which is well above the 5 kb packaging limit for AAV. Here we construct a < 5 kb FVIII‐AAV vector using murine FVIII cDNA and a strong liver‐specific albumin promoter. We assessed the efficacy of this vector using three different routes of administration, intraportal, intrasplenic and tail vein injection, in FVIII knockout (FVIII KO) mice. The peak level of FVIII observed was about 8% of normal mouse FVIII activity. Even at 9 months, post vector injection, 14 of 19 mice receiving FVIII‐AAV demonstrated phenotypic correction and roughly 2% FVIII activity. The transgene copy number ranged from 0.001 to 0.1 copies per cell, depending upon the somatic tissue. The potential for germline transmission of AAV was assayed in 34 pups obtained from five pairs of treated, phenotypically corrected adult hemophilic mice. Although the parents harbored the transgene in liver, spleen, and gonads, none of the 34 offspring was positive for the transgene, suggesting that the risk of inadvertent germline transmission is low.

Dual Vectors Expressing Murine Factor VIII Result in Sustained Correction of Hemophilia A Mice

Hemophilia A is a sex-linked disorder that results from a deficiency of functional factor VIII and is currently treated by protein replacement therapies. Within the past decade, gene therapy efforts have come to the forefront of novel therapeutics. In this work, a dual-vector approach was employed in which recombinant adeno-associated viral (rAAV) vectors expressing the heavy and light chains of the murine factor VIII gene were delivered either intramuscularly or intravenously to a mouse model of hemophilia A. From in vitro work, it was determined that coinfection with both vectors is required as heterodimerization of the heavy and light chains occurs intracellularly. In vivo, therapeutic levels of factor VIII expression were achieved throughout the duration of the study (22 weeks). Intravenous and intramuscular delivery resulted in a maximal average expression of 31.4 +/- 6.4 and 29 +/- 6.5% of normal murine factor VIII levels, respectively. Western blots of cryoprecipitate as well as immunostaining of injection sites with an anti-murine factor VIII light chain antibody also confirmed the expression of factor VIII. Because the murine form of the gene was used in the mouse model, less than 1 Bethesda unit of inhibitors was noted. This work demonstrates the feasibility of using rAAV vectors for the long-term treatment of hemophilia A.

Role of bone marrow transplantation for correcting hemophilia A in mice

To better understand cellular basis of hemophilia, cell types capable of producing FVIII need to be identified. We determined whether bone marrow (BM)-derived cells would produce cells capable of synthesizing and releasing FVIII by transplanting healthy mouse BM into hemophilia A mice. To track donor-derived cells, we used genetic reporters. Use of multiple coagulation assays demonstrated whether FVIII produced by discrete cell populations would correct hemophilia A. We found that animals receiving healthy BM cells survived bleeding challenge with correction of hemophilia, although donor BM-derived hepatocytes or endothelial cells were extremely rare, and these cells did not account for therapeutic benefits. By contrast, donor BM-derived mononuclear and mesenchymal stromal cells were more abundant and expressed FVIII mRNA as well as FVIII protein. Moreover, injection of healthy mouse Kupffer cells (liver macrophage/mononuclear cells), which predominantly originate from BM, or of healthy BM-derived mesenchymal stromal cells, protected hemophilia A mice from bleeding challenge with appearance of FVIII in blood. Therefore, BM transplantation corrected hemophilia A through donor-derived mononuclear cells and mesenchymal stromal cells. These insights into FVIII synthesis and production in alternative cell types will advance studies of pathophysiological mechanisms and therapeutic development in hemophilia A.