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Proceedings of the
8th International Symposium
on Canine and Feline Reproduction
ISCFR
June 22-25, 2016
Paris, France
In a joint meeting with the XIX EVSSAR Congress
Reprinted in IVIS with the permission of the ISCFR Organizers
Reprinted in IVIS with the permission of the Organizers
ISCFR VIII International Symposium on Canine and Feline Reproduction
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PARIS, FRANCE
JUNE 22-25, 2016
EVSSAR XIX
Engineering a gene silencing viral construct that targets the cat hypothalamus to induce
permanent sterility
Gregory A. Dissena, Alejandro Lomniczia, Kei Adachib, Thomas Chatkupt c, Beverly L. Davidsond,
Hiroyuki Nakaiab, and Sergio R. Ojedaa.
a
Division of Neuroscience, Oregon National Primate Research Center-Oregon Health & Science
University, Beaverton, OR 97006; bDepartment of Molecular & Medical Genetics, cDepartment of
Comparative Medicine, Oregon Health & Science University, Portland, OR 97239, USA;. dThe
Children’s Hospital of Philadelphia, Philadelphia, PA, 19104, USA
[email protected]
We are using a combined approach to permanently sterilize cats. This approach employs two
complementary methodologies: RNA interference (RNAi) to silence genes involved in the central
control of reproduction; and a virus-based gene therapy system intended to deliver RNAi
selectively to the hypothalamus (where these genes are expressed) via systemic administration of
modified viruses. We selected the hypothalamus because it contains neurons expressing Kiss1, a
gene essential for reproduction and fertility. We chose the non-pathogenic adeno-associated virus
(AAV) as a vector whose tropism could be modified to target the hypothalamus. We first used a
phage display library and a technique termed bio-panning to identify peptide sequences able to
reach the hypothalamus. We next modified the tropism of the AAV vector by inserting the most
promising peptide sequences into the viral capsid. Although we were able to re-target the AAV
vector from the liver to the hypothalamus, the level of hypothalamic transduction was not enough to
deliver RNAi at levels capable of reducing kisspeptin release and disrupt reproduction. To
overcome this limitation we turned to a novel approach termed AAV Barcode-Seq1 that is allowing
us to engineer and test hundreds of AAV mutants in a high throughput fashion using nextgeneration sequencing (NGS) technology. As a first step we performed a series of studies to
determine the prevalence of anti AAV antibodies in cat serum derived from feral, client-owned and
specific pathogen free (SPF) populations of cats (n = 94). The presence of antibodies targeting 11
common human serotypes of AAV was determined by ELISA. Overall, the cats with the highest
prevalence of antibodies to AAV were client owned cats. Feral and SPF cats had comparably low
levels of antibodies. Undetectable levels were found in immature SPF cats. Importantly, all cat
populations had very low levels of antibodies targeting the serotype AAV9, suggesting that AAV9
would be a good platform to engineer mutants able to target the hypothalamus. The validity of this
prediction was supported by the outcome of a study in which a DNA/RNA barcoded AAV library
containing 58 AAV strains, including 11 common AAV serotypes (1-11) and a diversity of AAV
mutants, was intravenously injected into two cats. Pharmacokinetic analysis of AAV clearance
from blood revealed that AAV9 variants remain longer in the circulation than other AAV species.
NGS analysis of a variety of peripheral tissues and brain regions allowed us to single out three
AAV9 variants devoid of liver tropism. We are now using these variants as a platform for studies
aimed at generating an AAV-based random octapeptide display library, which will be injected into
naive cats to identify AAV mutants able to reach the hypothalamus selectively with minimal vector
dissemination to other tissues.
[1] Adachi K, Enoki T, Kawano Y, Veraz M, Nakai H. Drawing a high-resolution functional map
of adeno-associated virus capsid by massively parallel sequencing. Nat Commun 2014; 5:3075 doi:
10.1038/ncomms4075 (2014)
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Proceedings of the International Symposium on Canine and Feline Reproduction, 2016 - Paris, France