Immunoadsorption enables successful rAAV5-mediated repeated hepatic gene delivery in nonhuman primates.

Adeno-associated virus (AAV)-based liver gene therapy has been shown to be clinically successful. However, the presence of circulating neutralizing antibodies (NABs) against AAV vector capsids remains a major challenge as it may prevent successful transduction of the target cells. Therefore, there is a need to develop strategies that would enable AAV-mediated gene delivery to patients with preexisting anti-AAV NABs. In the current study, the feasibility of using an immunoadsorption (IA) procedure for repeated, liver-targeted gene delivery in nonhuman primates was explored. The animals were administered IV with recombinant AAV5 (rAAV5) carrying the reporter gene human secreted embryonic alkaline phosphatase (hSEAP). Seven weeks after the first rAAV treatment, all of the animals were readministered with rAAV5 carrying the therapeutic hemophilia B gene human factor IX (hFIX). Half of the animals administered with rAAV5-hSEAP underwent IA prior to the second rAAV5 exposure. The transduction efficacies of rAAV5-hSEAP and rAAV5-hFIX were assessed by measuring the levels of hSEAP and hFIX proteins. Although no hFIX was detected after rAAV5-hFIX readministration without prior IA, all animals submitted to IA showed therapeutic levels of hFIX expression, and a threshold of anti-AAV5 NAB levels compatible with successful readministration was demonstrated. In summary, our data demonstrate that the use of a clinically applicable IA procedure enables successful readministration of an rAAV5-based gene transfer in a clinically relevant animal model. Finally, the analysis of anti-AAV NAB levels in human subjects submitted to IA confirmed the safety and efficacy of the procedure to reduce anti-AAV NABs. Furthermore, clinical translation was assessed using an immunoglobulin G assay as surrogate.

Mir-142-3p target sequences reduce transgene-directed immunogenicity following intramuscular adeno-associated virus 1 vector-mediated gene delivery.

BACKGROUND: Muscle represents an important tissue target for adeno-associated virus (AAV) vector-mediated gene transfer in muscular, metabolic or blood-related genetic disorders. However, several studies have demonstrated the appearance of immune responses against the transgene product after intramuscular AAV vector delivery that resulted in a limited efficacy of the treatment. Use of microRNAs that are specifically expressed in antigen-presenting cells (APCs) is a promising approach for avoiding those immune responses. Cellular mir-142-3p, which is APC-specific, is able to repress the translation of its target cellular transcripts by binding to a specific target sequences. METHODS: In the present study, we explored the potential of mir-142-3p specific target sequences with respect to reducing or abolishing immune responses directed against ovalbumin (OVA), a highly immunogenic protein, expressed as transgene and delivered by AAV1 vector administered intramuscularly. RESULTS: The occurrence of immune responses against OVA transgene following intramuscular delivery by AAV have been described previously and resulted in the loss of OVA protein expression. In the present study, we demonstrate that OVA protein expression was maintained when mir-142-3pT sequences were incorporated into the expression cassette. The sustained expression of OVA protein over time correlated with a reduced increase in anti-OVA antibody levels. Furthermore, no cellular infiltrates were observed in the muscle tissue when AAV1 vectors containing four or eight repeats of mir-142-3p target sequences after the OVA sequence were used. CONCLUSIONS: The rising humoral and cellular immune responses against OVA protein after intramuscular delivery can be efficiently reduced by the use of mir-142-3p target sequences.

Effect of arginine deficiency on arginine-dependent post-translational protein modifications in mice.

Transgenic mice that overexpress arginase-I in their small-intestinal enterocytes suffer from a pronounced, but selective decrease in circulating arginine levels during the suckling period, resulting in impaired growth and development of hair, muscle and immune system. In the present study, we tested the hypothesis that the arginine-deficiency phenotype is caused by arginine-specific post-translational modifications, namely, an increase in the degree of mono-ADP-ribosylation of proteins because of reduced competition by free arginine residues and/or an increase in protein-tyrosine nitration because of an increased O2- production by NO synthases in the presence of limiting amounts of arginine. Arginine ADP-ribosylation and tyrosine nitration of proteins in the affected organs were assayed by Western blot analysis, using specific anti-ADP-ribosylarginine and protein-nitrotyrosine antisera. The composition of the group of proteins that were preferentially arginine ADP-ribosylated or tyrosine-nitrated in the respective organs was strikingly similar. Arginine-deficient mice differed from their controls in a reduced ADP-ribosylation of a 130 kDa and a 65 kDa protein in skin and an increased protein nitration of an 83 kDa protein in bone marrow and a 250 kDa protein in spleen. Since only 20 % of the visualised proteins were differentially modified in a subset of the affected organs, our findings appear to rule out these prominent arginine-dependent post-translational protein modifications as mediators of the characteristic phenotype of severely arginine-deficient mice.

Arginine deficiency affects early B cell maturation and lymphoid organ development in transgenic mice.

Apart from its role in the synthesis of protein and nitric oxide (NO), and in ammonia detoxification, the amino acid arginine exerts an immunosupportive function. We have studied the role of arginine in immune defense mechanisms in the developing postnatal immune system. In suckling mice, arginine is produced in the small intestine. In F/A-2(+/+) transgenic mice, which overexpress arginase in their enterocytes, circulating and tissue arginine concentrations are reduced to 30-35% of controls. In these mice, the development and composition of the T cell compartment did not reveal abnormalities. However, in peripheral lymphoid organs and the small intestine, B cell cellularity and the number and size of Peyer's patches were drastically reduced, and serum IgM levels were significantly decreased. These phenotypes could be traced to an impaired transition from the pro- to pre-B cell stage in the bone marrow. Cytokine receptor levels in the bone marrow were normal. The development of the few peripheral B cells and their proliferative response after in vitro stimulation was normal. The disturbance in B cell maturation was dependent on decreased arginine levels, as this phenotype disappeared upon arginine supplementation and was not seen in NO synthase- or ornithine transcarbamoylase-deficient mice. We conclude that arginine deficiency impairs early B cell maturation.

Overexpression of arginase I in enterocytes of transgenic mice elicits a selective arginine deficiency and affects skin, muscle, and lymphoid development.

BACKGROUND: Arginine is required for the detoxification of ammonia and the synthesis of proteins, nitric oxide, agmatine, creatine, and polyamines, and it may promote lymphocyte function. In suckling mammals, arginine is synthesized in the enterocytes of the small intestine, but this capacity is lost after weaning. OBJECTIVE: We investigated the significance of intestinal arginine production for neonatal development in a murine model of chronic arginine deficiency. DESIGN: Two lines of transgenic mice that express different levels of arginase I in their enterocytes were analyzed. RESULTS: Both lines suffer from a selective but quantitatively different reduction in circulating arginine concentration. The degree of arginine deficiency correlated with the degree of retardation of hair and muscle growth and with the development of the lymphoid tissue, in particular Peyer's patches. Expression of arginase in all enterocytes was necessary to elicit this phenotype. Phenotypic abnormalities were reversed by daily injections of arginine but not of creatine. The expression level of the very arginine-rich skin protein trichohyalin was not affected in transgenic mice. Finally, nitric oxide synthase-deficient mice did not show any of the features of arginine deficiency. CONCLUSIONS: Enterocytes are important for maintaining arginine homeostasis in neonatal mice. Graded arginine deficiency causes graded impairment of skin, muscle, and lymphoid development. The effects of arginine deficiency are not mediated by impaired synthesis of creatine or by incomplete charging of arginyl-transfer RNA.