ObjectivePersistent adenoviral shedding in stools is known to occur past convalescence following acute adenoviral infections. We wished to establish the frequency with which adenoviruses may colonize the gut in normal human subjects.MethodsThe presence of adenoviral DNA in intestinal specimens obtained at surgery or autopsy was tested using a nested PCR method. The amplified adenoviral DNA sequences were compared to each other and to known adenoviral species. Lamina propria lymphocytes (LPLs) were isolated from the specimens and the adenoviral copy numbers in the CD4+ and CD8+ fractions were determined by quantitative PCR. Adenoviral gene expression was tested by amplification of adenoviral mRNA.ResultsIntestinal tissue from 21 of 58 donors and LPLs from 21 of 24 donors were positive for the presence of adenoviral DNA. The majority of the sequences could be assigned to adenoviral species E, although species B and C sequences were also common. Multiple sequences were often present in the same sample. Forty-one non-identical sequences were identified from 39 different tissue donors. Quantitative PCR for adenoviral DNA in CD4+ and CD8+ fractions of LPLs showed adenoviral DNA to be present in both cell types and ranged from a few hundred to several million copies per million cells on average. Active adenoviral gene expression as evidenced by the presence of adenoviral messenger RNA in intestinal lymphocytes was demonstrated in 9 of the 11 donors tested.ConclusionAdenoviral DNA is highly prevalent in lymphocytes from the gastro-intestinal tract indicating that adenoviruses may be part of the normal gut flora.
E1-deleted adenovirus vectors can be created from a wide variety of ape adenoviruses that can be rescued and propagated in HEK 293 cells. The prevalence of pre-existing antibodies that can neutralize these adenoviruses in human populations is low.
S26 factors in the development of the central nervous system (CNS) and severity of the neuropathology. We previously reported that early postnatal systemic delivery of the human aspartoacylase (hASPA) gene by recombinant adeno-associated virus (rAAV) to the CNS of a CD mouse model with neonatal death rescued lethality and partially restored motor function. Now in its 3 rd generation, our Canavan gene therapy completely reverses the disease phenotype in the CD KO mouse. For clinical translation to treat juvenile and adult patients, we sought to study and understand the age-limitations for Canavan disease gene therapy. We hypothesized that there is a point-of-noreturn, when gene replacement therapy alone is no longer sufficient to successfully alter the disease outcome. First, Nur7 mice were treated at post-natal (p) day 1 as the gold standard. In the next step, experimental groups were dosed at 6, 12, and 24 weeks of age with a dose 10-fold higher than that for neonates. Motor function was tested for all mice 4 weeks after treatment and subsequent intervals up to one year of age for direct comparison. As expected, the earlier mice were treated, the better the therapeutic outcome. To our surprise, juvenile mice at 6 weeks of age recovered completely within 4 weeks post-injection. Although mice treated at 3 months of age and older did not respond immediately within the first 4 weeks post-treatment, they eventually showed significant improvements over Nur7 mutant control mice. Of note, cognitive function testing revealed that treated mice recover cognitively before motor function improves; this was even true for late treatment time points. Furthermore, response to rAAVhASPA gene therapy was confirmed via MRS for N-acetyaspartate, MRI, and neuropathology. Finally, our preliminary data characterizing oligodendrocytes and myelin in the Nur7 mouse model shows rapid reconstitution of myelin in mice treated at 6 weeks, underlining the potential of our therapy for later treatment. Currently, we are working on identifying molecular mechanisms limiting complete disease rescue in adult Nur7 mice to further explore options in order to successfully treat older patients. Overall, our data demonstrate that rAAV mediated hASPA expression of our 3 rd generation gene therapy vector not only prevents but also rescues the clinical manifestation and pathology of the juvenile and adult model of Canavan disease at an unprecedented level, which might also have implications for other CNS disorders that require treatment in later stages of life.
S74 using PTPN11 knockdown in animal models will substantiate these observations and provide mechanistic insights into the roles of neurotrophic factor regulation in retina.
S276and comprehensive method to detect and characterise AAV-specific CD8 + T cells among PBMCs, we used Tetramer-Associated Magnetic Enrichment (TAME) to analyse the frequency and phenotype of AAVspecific CD8 + T cells by flow cytometry. To this end, we previously generated AAV-loaded tetramers using the UV-mediated peptide exchange technology. Results: We were able to detect AAV8 and AAV2 capsid-specific CD8 + T cells among PBMCs in all healthy donors tested (n>40), without any amplification, at frequencies ranging from 1.10 -6 to 1.10 -4 CD8 + T cells. Phenotypic assessment of the detected cells revealed a small proportion of memory CD45RO + cells, a population that expectedly could have emerged after primary infection with wild-type AAV. Though AAV-specific CD8 + T cells were detectable by TAME in all donors tested, only few of them (11/42) had a positive response when cellular responses in PBMCs were assessed by anti-AAV IFN-γ ELISpot assays. Interestingly, we registered higher ex vivo frequencies and more frequent positive ELISpot responses in HLA-B7 donors then in HLA-A2's. Evaluation of anti-AAV antibody and neutralizing factor titres in the sera of the same donors revealed no correlation between humoral and cellular responses, as has been previously described. To further the functional assessment of the detected cells, we sorted AAV8 capsid-specific CD8 + T cells after TAME and expanded human primary T cell lines. Purity of the cell lines was checked using tetramer staining. We succeeded in generating several functional AAV-specific CD8 + T cell lines that upregulated CD107 and secreted IFN-γ, TNF-α, Granzyme B and Perforin when faced with AAV8-loaded target cells. Interestingly, some cell lines seemed to display a lack of reactivity that could not be attributed to a generally compromised functionality, suggesting that a fine tuning of AAV-specific CD8 + T cells' activation might come into play, and is still under investigation. Conclusions and Perspectives: The dissimilarities observed ex vivo between humoral and cellular responses, as well as the different activation patterns registered in vitro highlight the need to gather multiple insights on capsid immunogenicity to better understand the onset of pre-existing anti-AAV immunity on recombinant AAV-based gene transfer and its impact on clinical outcome.
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