AAV vectors
A virus, selected technology, applied in the field of adeno-associated virus
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Embodiment 1
[0418] Embodiment 1: Novel AAV capsid
[0419]We performed AAV peptide display selections to identify AAVs with improved targeting to retinal cells. We used a library of AAV mutants displaying 7-mer random peptides (sometimes also 6-mers and 8-mers) at amino acid 587 of the AAV2 capsid protein VP1 (Perabo et al. (2003)” In vitro selection of viral vectors with modified tropism: the adeno-associated virus display, Molecular therapy: the journal of the American Society of Gene Therapy 8, 151-157). At either end of both ends (N-terminal and C-terminal), the inserted peptide is flanked by There are 2-3 additional "flexible" amino acids (such as alanine). Amino acid position 587 is located at the tip of the peak on the threefold axis of symmetry of the capsid (Xie et al. (2002) "The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy”Proceedings of the National Academy of Sciences of the United States of America 99, 10405-10410), and within the hepa...
Embodiment 2
[0425] Example 2: Biological testing of novel AAV capsids
[0426] Insert synthetic DNA oligonucleotides encoding selected peptide variants flanking either end at the Asel / Mlul restriction sites of the AAV trans plasmid pRC99 (expressing the AAV2 Rep and Cap genes) Each has 2-3 alanines (Nickiin et al. (2001) "Efficient and selective AAV2-mediated gene transferdirected to human vascular endothelial cells, Molecular therapy: the journal of the American Society of Gene Therapy 4, 174-181, and Girod et al. al. (1999) "Geneticcapsid modifications allow efficient re-targeting of adeno-associated virustype 2", Nature medicine 5, 1052-1056), leading to the generation of the AAV2 VP1-3 open reading frame with the expected peptide insertion located at the site corresponding to VP1 The position of the 587th amino acid. These modified pRC99 plasmids are used to produce AAV particles containing novel AAV capsid variants for biological testing. The production of AAV is carried out by (Mich...
Embodiment 3
[0432] Example 3: Retina cross section
[0433] To show that the AAV vectors described herein indeed transduce photoreceptors, the AAV2 "GLSPPTR" capsid has been applied intravitreally to live mice. Specifically, a single intravitreal injection of 1 μl (containing approximately 2×10E9 vector genomes) of ss-mSWS-eGFP virus suspension packaged with AAV2 "GLSPPTR" capsid (see also Example 2) was applied intravitreally at 10 weeks of age. C57-BL6J mice. AAV-ss-mSWS-eGFP drives the expression of eGFP under the control of the mouse SWS promoter. Expression of GFP was measured after 6 weeks. For this, confocal scanning microscope images are acquired from the photoreceptor region of the eye. Such as Figure 12 As shown, eGFP can be detected in retinal cross-sections of cone photoreceptors. Here, a strong anti-eGFP immune signal (gray) in cone photoreceptors is observed. Double labeling quantification of the cone cell marker cone inhibin showed that 78.7±3.1% (n=4) of the cone in...
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