Patricia Berthelette

Correction of Enzymatic and Lysosomal Storage Defects in Fabry Mice by Adenovirus-Mediated Gene Transfer

Fabry disease is a recessive, X-linked disorder caused by a deficiency of the lysosomal hydrolase alpha-galactosidase A. Deficiency of this enzyme results in progressive deposition of the glycosphingolipid globotriaosylceramide (GL-3) in the vascular lysosomes, with resultant distension of the organelle. The demonstration of a secretory pathway for lysosomal enzymes and their subsequent recapture by distant cells through the mannose 6-phosphate receptor pathway has provided a rationale for somatic gene therapy of lysosomal storage disorders. Toward this end, recombinant adenoviral vectors encoding human alpha-galactosidase A (Ad2/CEHalpha-Gal, Ad2/CMVHIalpha-Gal) were constructed and injected intravenously into Fabry knockout mice. Administration of Ad2/CEHalpha-Gal to the Fabry mice resulted in an elevation of alpha-galactosidase A activity in all tissues, including the liver, lung, kidney, heart, spleen, and muscle, to levels above those observed in normal animals. However, enzymatic expression declined rapidly such that by 12 weeks, only 10% of the activity observed on day 3 remained. Alpha-galactosidase A detected in the plasma of injected animals was in a form that was internalized by Fabry fibroblasts grown in culture. Such internalization occurred via the mannose 6-phosphate receptors. Importantly, concomitant with the increase in enzyme activity was a significant reduction in GL-3 content in all tissues to near normal levels for up to 6 months posttreatment. However, as expression of alpha-galactosidase A declined, low levels of GL-3 reaccumulated in some of the tissues at 6 months. For protracted treatment, we showed that readministration of recombinant adenovirus vectors could be facilitated by transient immunosuppression using a monoclonal antibody against CD40 ligand (MR1). Together, these data demonstrate that the defects in alpha-galactosidase A activity and lysosomal storage of GL-3 in Fabry mice can be corrected by adenovirus-mediated gene transfer. This suggests that gene replacement therapy represents a viable approach for the treatment of Fabry disease and potentially other lysosomal storage disorders.

Local gene delivery of heme oxygenase-1 by adeno-associated virus into osteoarthritic mouse joints exhibiting synovial oxidative stress

OBJECTIVE To evaluate the role of synovial oxidative stress on joint pathology in a spontaneous mouse model of osteoarthritis (OA) by intra-articular (IA) delivery of recombinant adeno-associated virus (rAAV) expressing anti-oxidant protein heme oxygenase-1 (HO-1). METHODS Joint transduction by rAAV vectors was evaluated with serotype 1, 2, 5 and 8 capsids carrying LacZ gene administered by IA injections into STR/ort mice. Transduced cell types were identified by β-galactosidase staining in sectioned joints. Effect of oxidative stress on AAV transduction of primary synoviocytes in vitro was quantitated by fluorescence-activated cell sorting (FACS) analysis. In vivo, the efficacy of rAAV1/HO-1 was tested by IA administration into STR/ort mice followed by histopathological scoring of cartilage. Levels of 3-nitrotyrosine (3-NT) and HO-1 were assessed by immunohistochemistry (IHC) of joint sections. RESULTS Administration of a rAAV1 based vector into OA mouse joints resulted in transduction of the synovium, joint capsule, adipocytes and skeletal muscle while none of the serotypes showed significant cartilage transduction. All OA joints exhibited significantly elevated levels of oxidative stress marker, 3-NT, in the synovium compared to OA-resistant CBA-strain of mice. In vitro studies demonstrated that AAV transgene expression in primary synoviocytes was augmented by oxidative stress induced by H(2)O(2) and that a rAAV expressing HO-1 reduced the levels of oxidative stress. In vivo, HO-1 was increased in the synovium of STR/ort mice. However, delivery of rAAV1/HO-1 into OA joints did not reduce cartilage degradation. CONCLUSIONS AAV-mediated HO-1 delivery into OA joints during active disease was not sufficient to improve cartilage pathology in this model.

Overexpression of cystatin C in synovium does not reduce synovitis or cartilage degradation in established osteoarthritis

IntroductionCathepsin K (catK) expression is increased in cartilage, bone and synovium during osteoarthritis (OA). To study the role of catK expression and elevated cathepsin activity in the synovium on cartilage destruction in established OA, we overexpressed cystatin C (cysC), a natural cysteine protease inhibitor, in the synovium of rabbit OA joints.MethodsThe ability of cysC to inhibit activity of cathepsins in rabbit OA synovium lysates was tested in vitro using protease activity assay. In vivo, the tissue localization of recombinant adeno-associated virus (rAAV) with LacZ gene after intra-articular injection was determined by β-galactosidase staining of rabbit joints 4 weeks later. To inhibit cathepsin activity in the synovium, a rAAV2-encoding cysC was delivered intra-articularly into rabbit joints 4 weeks after OA was induced by anterior cruciate ligament transection (ACLT). Seven weeks postinjection, endogenous catK and cysC levels as well as the vector-derived cysC expression in the synovium of normal and OA joints were examined by RNA quantification. Synovial cathepsin activity and catK, catB and catL protein levels were determined by activity and Western blot analyses, respectively. Synovitis and cartilage degradation were evaluated by histopathological scoring.ResultsIn vitro, the ability of cysC to efficiently inhibit activity of purified catK and OA-induced cathepsins in rabbit synovial lysates was demonstrated. In vivo, the intra-articular delivery of rAAV2/LacZ showed transduction of mostly synovium. Induction of OA in rabbit joints resulted in fourfold increase in catK mRNA compared to sham controls while no change was detected in endogenous cysC mRNA levels in the synovium. Protein levels for catK, catB and catL were also increased in the synovium with a concomitant fourfold increase in cathepsin activity. Joints treated with rAAV2/cysC showed both detection of vector genomes and vector-derived cysC transcripts in the synovium. Production of functional cysC by the vector was demonstrated by complete block of cathepsin activity in the synovium. However, this did not decrease synovitis, bone sclerosis or progression of cartilage degradation.ConclusionsIncreased production of natural cathepsin inhibitor, cysC, in OA synovium does not alleviate synovitis or cartilage pathology during a preexisting OA.

The impact of minimally oversized adeno-associated viral vectors encoding human factor VIII on vector potency in vivo

Recombinant adeno-associated viral (rAAV) vectors containing oversized genomes provide transgene expression despite low efficiency packaging of complete genomes. Here, we characterized the properties of oversized rAAV2/8 vectors (up to 5.4 kb) encoding human factor VIII (FVIII) under the transcriptional control of three liver promoters. All vectors provided sustained production of active FVIII in mice for 7 months and contained comparable levels of vector genomes and complete expression cassettes in liver. Therefore, for the 5.4 kb genome size range, a strong expression cassette was more important for FVIII production than the vector genome size. To evaluate the potency of slightly oversized vectors, a 5.1 kb AAVrh8R/FVIII vector was compared to a 4.6 kb (wild-type size) vector with an identical expression cassette (but containing a smaller C1-domain deleted FVIII) for 3 months in mice. The 5.1 kb vector had twofold to threefold lower levels of plasma FVIII protein and liver vector genomes than that obtained with the 4.6 kb vector. Vector genomes for both vectors persisted equally and existed primarily as high molecular weight concatemeric circular forms in liver. Taken together, these results indicate that the slightly oversized vectors containing heterogeneously packaged vector genomes generated a functional transgene product but exhibited a twofold to threefold lower in vivo potency.

Hyaluronic acid synthase-2 gene transfer into the joints of Beagles by use of recombinant adeno-associated viral vectors

OBJECTIVE To evaluate gene transfer of recombinant adeno-associated viral (rAAV) vectors with AAV2 or AAV5 capsid and encoding hyaluronic acid (HA) synthase-2 (HAS2) into joints of healthy dogs. ANIMALS 22 purpose-bred Beagles. PROCEDURES Plasmid expression cassettes encoding canine HAS2 (cHAS2) were assessed in vitro for concentration and molecular size of secreted HA. Thereafter, rAAV2-cHAS2 vectors at 3 concentrations and rAAV5-cHAS2 vectors at 1 concentration were each administered intra-articularly into the left stifle joint of 5 dogs; 2 dogs received PBS solution instead. Synovial fluid HA concentration and serum and synovial fluid titers of neutralizing antibodies against AAV capsids were measured at various points. Dogs were euthanized 28 days after treatment, and cartilage and synovium samples were collected for vector DNA and mRNA quantification and histologic examination. RESULTS Cell transfection with plasmids encoding cHAS2 resulted in an increase in production and secretion of HA in vitro. In vivo, the rAAV5-cHAS2 vector yielded uniform genome transfer and cHAS2 expression in collected synovium and cartilage samples. In contrast, rAAV2-cHAS2 vectors were detected inconsistently in synovium and cartilage samples and failed to produce clear dose-related responses. Histologic examination revealed minimal synovial inflammation in joints injected with rAAV vectors. Neutralizing antibodies against AAV capsids were detected in serum and synovial fluid samples from all vector-treated dogs. CONCLUSIONS AND CLINICAL RELEVANCE rAAV5-mediated transfer of the gene for cHAS2 into healthy joints of dogs by intra-articular injection appeared safe and resulted in vector-derived cHAS2 production by synoviocytes and chondrocytes. Whether this treatment may increase HA production by synoviocytes and chondrocytes in osteoarthritic joints remains to be determined.

92. Evaluation of Producer Cell Line Platform for Production of Oversized AAV-FVIII Vectors

Recombinant adeno-associated virus (rAAV) vectors are being evaluated as gene delivery vehicles in several clinical trials. The 4.7 kb wild-type (WT) size genome of AAV presents a challenge for incorporating larger transgenes with incomplete vector genome (vg) packaging being a frequent outcome. To test the feasibility of producing oversized rAAV production using the producer cell line (PCL) method, we generated slightly oversized rAAV vectors (harboring 5.1 or 5.4 kb sized vgs) containing a liver-restricted promoter (mTTR) and a codon-optimized cDNA encoding human B-domain deleted FVIII (FVIIIco). Genomes were packaged into the AAVrh8R serotype vector using the PCL process and compared to matched vectors generated via the triple transfection (TXN) method. Vectors were then characterized for production yields, integrity of packaged genomes and homogeneity. The data showed that the PCL platform was able to produce oversized AAV vectors at levels that were 10- to 100-fold higher than the TXN process with yields greater than 100,000 vg/cell. The PCLs were stable with consistent production maintained up to 60 passages. Southern and dot blot analyses of the packaged genomes demonstrated encapsidation of genomes larger than 4.7 kb in the PCL generated vector while the majority of genomes packaged via the TXN method were 4.7 kb in size. Furthermore, the PCL process generated more vector DNA-containing particles and less packaging of non-vector DNA. Testing the PCL generated vectors in the hemophilia A knock-out (KO) mouse model showed a 2-fold higher plasma FVIII activity (Coatest) and vg copies in the livers than obtained with the vectors made by the TXN process. In summary, the PCL production process generated higher yields of oversized rAAV/FVIIIco vectors as well as higher quality vectors than the TXN method. Hence, the PCL platform may be used for producing greater quality oversized rAAV vectors at levels that can meet the needs for clinical studies.

387. Transfer of Hyaluronic Acid Synthase-2 Gene into Canine Joints Using Adeno-Associated Viral Vectors

Osteoarthritis (OA) is one of the more common causes of lameness in dogs and is estimated to affect approximately 20% of dogs >1 year old. OA is a progressive and degenerative disease that results in pain, inflammation and reduced joint mobility. Novel, safe and efficacious therapies that improve joint lubrication and reduce the extent of inflammation and pain are potential solutions for the management of canine OA. Here, we evaluated the efficiency of gene transfer to canine joint by intra-articular injections of recombinant adeno-associated virus (rAAV) vectors encoding therapeutic genes. Specifically, we generated rAAV vectors (either AAV2 and AAV5 serotypes vectors) encoding canine codon-optimized hyaluronic acid (HA) synthase-2 (HAS2) to provide local and continuous synthesis of HA in the joint. Twenty-two adult healthy dogs that were seronegative for anti-AAV2 and -AAV5 antibodies were injected intra-articularly with rAAV2/HAS2 (1, 5 and 10×1011 vg/joint), rAAV5/HAS2 (5×1011 vg/joint) or PBS (control). No adverse clinical signs were observed following the 28-day study period. Histopathological analysis showed minimal synovial inflammation in the joints of dogs treated with rAAV5/HAS2 and no significant changes in the rAAV2/HAS2 treated dogs. Vector genomes (VG) were detected in the synovium of all the rAAV-treated joints and in the majority of cartilage samples tested. The rAAV5/HAS2 vector resulted in findings of higher and more consistent detection of VG and transcripts compared to rAAV2/HAS2 in both the synovial and cartilage samples. Preliminary analysis also showed a trend towards increased HA levels in the synovial fluid of the treated joints. In summary, our study demonstrated that rAAV2 and rAAV5-mediated gene transfer to canine joint tissues is feasible and that both vectors present an acceptable safety profile following a single intra-articular injection.

247. Characterization of Oversized rAAV Vectors for Human FVIII Gene Transfer

Severe hemophilia A patients with 4 years and consequent reduction or elimination of the need for frequent factor infusions. Similar rAAV-mediated gene transfer for hemophilia A patients is challenging due to the size of the FVIII cDNA that when combined with the necessary transcriptional elements exceeds the rAAV packaging limit of 4.7 kb. To test the feasibility of rAAV-mediated gene therapy for hemophilia A, we generated several oversized FVIII expression cassettes of different lengths and containing human B-domain deleted FVIII cDNA. These constructs were used to produce rAAV vectors by triple transfection and virus quality and yield were analyzed. Characterization of the packaged vector genomes (VGs) by Southern blot showed that the majority of genomes were 4.7 kb in size (with some being larger) and that the vector yield was affected by VG size. The vectors were also administered intravenously into C57BL/6 mice to test in vivo potency. Plasma FVIII levels were measured for 7 months post-treatment and at the end of the study, levels of FVIII mRNA and liver VG copies were quantitated. The data showed intact expression cassettes and comparable number of VG copies in liver among the vectors. Also, the higher liver mRNA levels correlated with higher plasma FVIII levels indicating that expression levels were a limiting factor. Lastly, the rAAV vector ranking was similar to ranking of plasmid expression cassettes (hydrodynamic injections) in vivo. These studies confirmed the viability of oversized rAAV vectors as an approach for the treatment of severe hemophilia A.