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      Durable contraception in the female domestic cat using viral-vectored delivery of a feline anti-Müllerian hormone transgene

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          Abstract

          Eighty percent of the estimated 600 million domestic cats in the world are free-roaming. These cats typically experience suboptimal welfare and inflict high levels of predation on wildlife. Additionally, euthanasia of healthy animals in overpopulated shelters raises ethical considerations. While surgical sterilization is the mainstay of pet population control, there is a need for efficient, safe, and cost-effective permanent contraception alternatives. Herein, we report evidence that a single intramuscular treatment with an adeno-associated viral vector delivering an anti-Müllerian hormone transgene produces long-term contraception in the domestic cat. Treated females are followed for over two years, during which transgene expression, anti-transgene antibodies, and reproductive hormones are monitored. Mating behavior and reproductive success are measured during two mating studies. Here we show that ectopic expression of anti-Müllerian hormone does not impair sex steroids nor estrous cycling, but prevents breeding-induced ovulation, resulting in safe and durable contraception in the female domestic cat.

          Abstract

          This study demonstrates the safety and long-term efficacy of a single-dose, injectable contraceptive in female domestic cats. Treated females remained contracepted for over two years, and did not ovulate or produce kittens when paired with males.

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          Most cited references61

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          Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction

          Base editors enable targeted single-nucleotide conversion in genomic DNA. Here we show that expression levels are a bottleneck in base editing efficiency. We optimize cytidine (BE4) and adenine (ABE7.10) base editors by modification of nuclear localization signals and codon usage, and ancestral reconstruction of the deaminase component. The resulting BE4max, AncBE4max, and ABEmax editors correct pathogenic SNPs with substantially increased efficiency in a variety of mammalian cell types.
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            Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy.

            Tissues from rhesus monkeys were screened by PCR for the presence of sequences homologous to known adeno-associated virus (AAV) serotypes 1-6. DNA spanning entire rep-cap ORFs from two novel AAVs, called AAV7 and AAV8, were isolated. Sequence comparisons among these and previously described AAVs revealed the greatest divergence in capsid proteins. AAV7 and AAV8 were not neutralized by heterologous antisera raised to the other serotypes. Neutralizing antibodies to AAV7 and AAV8 were rare in human serum and, when present, were low in activity. Vectors formed with capsids from AAV7 and AAV8 were generated by using rep and inverted terminal repeats (ITRs) from AAV2 and were compared with similarly constructed vectors made from capsids of AAV1, AAV2, and AAV5. Murine models of skeletal muscle and liver-directed gene transfer were used to evaluate relative vector performance. AAV7 vectors demonstrated efficiencies of transgene expression in skeletal muscle equivalent to that observed with AAV1, the most efficient known serotype for this application. In liver, transgene expression was 10- to 100-fold higher with AAV8 than observed with other serotypes. This improved efficiency correlated with increased persistence of vector DNA and higher number of transduced hepatocytes. The efficiency of AAV8 vector for liver-directed gene transfer of factor IX was not impacted by preimmunization with the other AAV serotypes. Vectors based on these novel, nonhuman primate AAVs should be considered for human gene therapy because of low reactivity to antibodies directed to human AAVs and because gene transfer efficiency in muscle was similar to that obtained with the best known serotype, whereas, in liver, gene transfer was substantially higher than previously described.
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              Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection.

              This study examines transgene expression and biodistribution of adeno-associated virus (AAV) pseudotyped 1-9 after tail vein (TV) injection in male mice. Using a cytomegalovirus (CMV)-luciferase transgene, the time-course of expression in each animal was tracked throughout the experiment. The animals were imaged at 7, 14, 29, 56, and 100 days after the TV injection. The total number of photons emitted from each animal was recorded, allowing examination of expression level and kinetics for each pseudotyped virus. The bioluminescence imaging revealed three expression levels (i) low-expression group, AAV2, 3, 4, and 5; (ii) moderate-expression group, AAV1, 6, and 8; and (iii) high-expression group, AAV7 and 9. In addition, imaging revealed two classes of kinetics (i) rapid-onset, for AAV1, 6, 7, 8, and 9; and (ii) slow-onset, for AAV2, 3, 4, and 5. We next evaluated protein expression and viral genome copy numbers in dissected tissues. AAV9 had the best viral genome distribution and highest protein levels. The AAV7 protein and genome copy numbers were comparable to those of AAV9 in the liver. Most surprisingly, AAV4 showed the greatest number of genome copies in lung and kidney, and a high copy number in the heart. AAV6 expression was observed in the heart, liver, and skeletal muscle, and the genome distribution corroborated these observations.
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                Author and article information

                Contributors
                bill.swanson@cincinnatizoo.org
                dpepin@mgh.harvard.edu
                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Publishing Group UK (London )
                2041-1723
                6 June 2023
                6 June 2023
                2023
                : 14
                : 3140
                Affiliations
                [1 ]GRID grid.446612.3, ISNI 0000 0000 9486 2488, Center for Conservation and Research of Endangered Wildlife (CREW), , Cincinnati Zoo & Botanical Garden, ; Cincinnati, OH USA
                [2 ]GRID grid.38142.3c, ISNI 000000041936754X, Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Department of Surgery, , Harvard Medical School, ; Boston, MA USA
                [3 ]GRID grid.168645.8, ISNI 0000 0001 0742 0364, Horae Gene Therapy Center, , University of Massachusetts Chan Medical School, ; Worcester, MA USA
                Author information
                http://orcid.org/0000-0003-0857-6891
                http://orcid.org/0000-0002-5443-0175
                http://orcid.org/0000-0002-7314-2574
                http://orcid.org/0000-0002-8611-4767
                http://orcid.org/0000-0002-4122-2406
                http://orcid.org/0000-0003-0097-9012
                http://orcid.org/0000-0001-9079-2360
                http://orcid.org/0000-0003-4067-0319
                http://orcid.org/0000-0003-2046-6708
                Article
                38721
                10.1038/s41467-023-38721-0
                10244415
                37280258
                5de40f61-bbd2-4dde-88d3-f1ede914ddf4
                © The Author(s) 2023

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 27 June 2022
                : 10 May 2023
                Funding
                Funded by: FundRef https://doi.org/10.13039/100006111, Michelson Prize and Grants (Michelson Prize & Grants);
                Award ID: 2014D004314
                Award ID: 2014D004314
                Award ID: 2014D004314
                Award ID: 2014D004314
                Award ID: 2014D004314
                Award Recipient :
                Funded by: FundRef https://doi.org/10.13039/100005294, Massachusetts General Hospital (MGH);
                Award ID: 2021A012059
                Award Recipient :
                Funded by: Michelson Prize and Grants (Michelson Prize & Grants)
                Funded by: Michelson Prize and Grants (Michelson Prize & Grants)
                Funded by: Michelson Prize and Grants (Michelson Prize & Grants)
                Funded by: Michelson Prize and Grants (Michelson Prize & Grants)
                Categories
                Article
                Custom metadata
                © Springer Nature Limited 2023

                Uncategorized
                gene therapy,genetic engineering,reproductive biology,target validation
                Uncategorized
                gene therapy, genetic engineering, reproductive biology, target validation

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