Adeno-associated viral vectors for treatment of Niemann-Pick type C disease
A technology of viral vectors and vectors, applied in the direction of viruses/bacteriophages, using vectors to introduce foreign genetic material, viruses, etc.
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Embodiment 1
[0387] Example 1. Combinatorial engineering and selection of AAV vectors for antibody evasion
[0388] Antibody evasion AAV mutants were prepared according to the following method. The first step involves the identification of conformational 3D epitopes on the capsid surface of the AAV capsid protein, eg, using cryo-electron microscopy. Selected residues within the antigenic motif are then subjected to mutagenesis using degenerate primers, wherein each codon is replaced by the nucleotide NNK and the gene fragments are brought together by Gibson assembly and / or multi-step PCR. Genes encoding capsid protein subunits containing a degenerate library of mutated antigenic motifs were cloned into the wild-type AAV genome in place of the original Cap-encoding DNA sequence, resulting in a plasmid library. The plasmid library was then transfected with an adenovirus helper plasmid into 293 producer cell lines to generate an AAV capsid protein subunit library, which was then subjected to...
Embodiment 2
[0394] Example 2: Manufacturability of recombinant AAV vectors
[0395] To determine whether the various recombinant AAVs identified in Example 1 can be manufactured in a large-scale system, AAVs were produced according to standard methods and the yields were compared to those of wild-type AAV vectors.
[0396] AAVs were produced in HEK293 cells according to standard triple transfection protocols. Briefly, using (i) the wild-type AAV9 capsid protein subunit sequence STRD.101 capsid protein subunit variant sequence (SEQ ID NO: 180) or the STRD.102 capsid protein subunit variant sequence ( SEQ ID NO: 175), (ii) a plasmid containing the 5'ITR, transgene, and 3'ITR sequences, and (ii) a plasmid containing an accessory gene necessary for AAV production, transfected the cells. In the self-complementary construct, two different transgenes were used for each capsid protein subunit. Cells were then lysed and virions were purified using affinity columns, CsCl density ultracentrifugati...
Embodiment 3
[0398] Example 3: In vitro transduction using recombinant AAV viral vectors
[0399] To confirm whether the recombinant AAV vectors of Example 1 were generally infectious and capable of transducing cells in culture, various AAV vectors were prepared according to standard protocols.
[0400] Recombinant AAVs were tested for infectivity using a standard TCID50 assay. Briefly, HeLaRC32 cells were infected with recombinant AAV particles in the presence of adenovirus (Ad5) at doses spanning 5 orders of magnitude. After 72 hours, DNA was extracted and vector genome replication was quantified by qPCR.
[0401] The ratio of particles to infectivity was calculated to determine infectivity. like image 3 As shown, the infectivity ratio of the AAV-STRD.101 vector is lower compared to the infectivity of wild-type AAV9. AAV-STRD.101 is more infectious than wild-type AAV9 because the lower infectivity ratio translates to higher potency.
[0402] Separately, infectivity was also determi...
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