468 glp compliant non clinical safety and biodistribution of a recombinant aav2 8 vector administered intravenously for treatment of mucopolysaccharidosis type vi

Mucopolysaccharidosis VI (MPS VI) is a lysosomal storage disorder caused by deficiency of the enzyme arylsulfatase B (ARSB), which results in widespread accumulation and excretion of toxic glycosaminoglycans. We recently developed a successful gene therapy approach based on a single systemic administration of AAV2/8 that targets liver of MPS VI animal models. In view of a gene therapy clinical trial for MPS VI, we performed GLP-compliant non-clinical studies to assess the safety and biodistribution of AAV2/8. TBG. hARSB, a recombinant AAV2/8 vector encoding human ARSB (hARSB) under the control of the thyroxine-binding globulin promoter (TBG). We used transgenic C57/BL6-TgARSBC91S mice that overexpress an inactive hARSB C91S mutant and are thus immune tolerant to hARSB. Mice were treated with either AAV2/8.TBG. hARSB or the vehicle alone, as control. Toxicity was evaluated on day 15 (D15) and 180 (D180) after systemic injection of 2×1013 gc/kg, which is 10X the highest dose proposed for the clinical study [20males(M)+20females(F)/treatment/timepoint]. No mortality, abnormal clinical signs and alteration in body weight, body temperature and food intake were observed through the study. Similarly, no clinically relevant changes in blood chemistry and hematology were found in treated mice compared to controls. Histopathology revealed thyroid epithelial hypertrophy in AAV-treated mice. AAV2/8.TBG. hARSB biodistribution and expression was evaluated on D15 and D180 at the dose of 2×1012 gc/kg, which is 1X the highest dose proposed for the clinical study (5M+5F/treatment/timepoint). Although vector DNA was present in all organs on D15, it was sequestered mainly in liver at levels at least 3 logs higher than those found in other organs. Vector DNA declined on D180, but remained high in liver. Accordingly, hARSB was mainly expressed stably in liver, supporting TBG tissue specificity. Vector DNA was found in gonads of both sexes at 3 logs lower than in liver. A robust reduction of vector DNA was observed on D180. A supportive study conducted in male rabbits showed that vector shedding in semen was only transient, which suggests that the risk of inadvertent germline transmission of AAV2/8. TBG.hARSB is minimal at least in male animals. An in situ hybridization study is ongoing in ovaries to elucidate AAV localization. Finally, AAV DNA was only transiently present in plasma, urine and stools of mice (up to D37, D2 and D14, respectively), which minimizes the potential risk associated with transmission to third parties and/or the environment. In conclusion, these studies show a safe profile of intravenous administrations of AAV2/8. TBG.hARSB and pave the way for the phase I/II clinical trial

372. Prevalence of Anti-AAV8 Neutralizing Antibodies and ARSB Cross-Reactive Immunologic Material in MPS VI Patients Candidates for a Gene Therapy Trial

Recombinant vectors based on adeno-associated virus serotype 8 (AAV8) have been successfully used in the clinic and hold great promise for liver-directed gene therapy. Pre-existing immunity against AAV8 or the development of antibodies against the therapeutic transgene product might negatively affect the outcomes of gene therapy. In the prospect of an AAV8-mediated, liver-directed gene therapy clinical trial for Mucopolysaccharidosis VI (MPS VI), a lysosomal storage disorder due to arylsulfatase B (ARSB) deficiency, we investigated in a multiethnic cohort of MPS VI patients the prevalence of neutralizing antibodies (Nab) to AAV8 and the presence of ARSB cross-reactive immunologic material (CRIM), which will either affect the efficacy of gene transfer or the duration of phenotypic correction. Thirty-six MPS VI subjects included in the study harbored 45 (62.5%) missense, 13 (18%) nonsense, 9 (12.5%) frameshift (2 insertions and 7 deletions), and 5 (7%) splicing ARSB mutations. To the best of our knowledge, four mutations had not been previously described. These include: one missense (c.1178 A>G p.H393R) and three frameshift mutations [883-884duplTT (p.F295FfsX42), c.1036delG (p.E346SfsX11), c.1475delC (pP492LfsX80)] predicted to result in truncated proteins. The detection of ARSB protein in twenty-four patients out of 34 (71%) was predicted by the type of mutations. Pre-existing Nab to AAV8 were undetectable in 19/33 (58%) analyzed patients. Twelve out of 31 patients (39%) tested were both negative for Nab to AAV8 and CRIM-positive. In conclusion, this study allows estimating the number of MPS VI patients eligible for a gene therapy trial by intravenous injections of AAV8

An Anti-CD2 Monoclonal Antibody That Both Inhibits and Stimulates T Cell Activation Recognizes a Subregion of CD2 Distinct from Known Ligand-Binding Sites

The T lymphocyte glycoprotein CD2 appears to have an important role in human T cell development and activation. A novel anti-CD2 monoclonal antibody, designated UMCD2, was shown to block E rosetting, and therefore was defined as recognizing the T111 ligand-binding epitope. Binding of UMCD2 to T cells and thymocytes was blocked by several, but not all, anti-T111 antibodies, suggesting that the T111 eptope consists of more than one subepitope. In functional studies, the combination of UMCD2 plus anti-T113 was mitogenic for T cells; in some individuals, the level of activation was as high as that seen for the combination of anti-T112 plus anti-T113. However, when UMCD2 was added to other stimuli mitogenic for T lymphocytes, such as IL-2 or anti-CD3-Sepharose, it inhibited T cell responses. Although the combination of UMCD2 and anti-T113 induced an increase in cytoplasmic free calcium, the inhibitory activities of UMCD2 were not accompanied by effects on calcium fluxes. A panel of previously characterized CD2 mutants was then analyzed for binding of UMCD2 and other anti-CD2 monoclonals. Surprisingly, UMCD2 bound to all mutants tested, although the other anti-CD2 antibodies with specificity for the ligand-binding region of CD2 each failed to bind to one or more mutants. These data suggest that binding of antibody to a particular CD2 epitope can have opposite effects on the state of T cell activation, depending on the costimulus. Moreover, inhibitory effects mediated through CD2 may use a signaling mechanism distinct from that used in CD2 pathway activation. Of particular interest, the portion of the CD2 ligand-binding region recognized by UMCD2 is distinct from areas of the CD2 molecule that have previously been studied.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30600/1/0000237.pd

A Clinical Trial of Gene Therapy For Mucopolysaccharidosis Vi, A Severe Lysosomal Storage Disorder (Meusix)

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