Compositions and methods for in vivo nuclease-mediated gene targeting for the treatment of genetic disorders

A system for treating a genetic disorder, the system comprising:

• (a) a gene editing AAV comprising an AAV capsid and a first vector genome comprising a 5′ ITR, a sequence encoding a meganuclease that targets PCSK9 under control of regulatory sequences that direct expression of the meganuclease in a target cell comprising a PCSK9 gene, and a 3′ ITR;
• and
• (b) a donor AAV vector comprising an AAV capsid and a second vector genome comprising:
• a 5′ITR, a 5′ homology directed recombination (HDR) arm, a transgene and regulatory sequences that direct expression of the transgene in the target cell, a 3′ HDR arm, and a 3′ ITR.

A system for treating a genetic disorder, the system comprising:

• (a) a gene editing AAV comprising an AAV capsid and a first vector genome comprising a 5′ ITR, a 5′ nuclear localization signal (NLS), a sequence encoding a Cas9 and regulatory sequences that direct expression of the saCas9 in a target cell comprising the PCSK9 gene, a 3′ NLS, and a 3′ ITR;
• and
• (b) a donor AAV vector comprising an AAV capsid and a second vector genome comprising:
• a 5′ITR, a 5′ homology directed recombination (HDR) arm, a transgene and regulatory sequences that direct expression of the transgene in the target cell, a 3′ HDR arm, a U6 promoter, a sgRNA comprising at least 20 nucleotides that specifically bind to a target site in the PCSK9 gene, said target site being 5′ to a protospacer-adjacent motif (PAM) that is specifically recognized by the Cas9, and a 3′ ITR.

A system for treating a genetic disorder, the system comprising:

• (a) a gene editing AAV vector comprising an AAV capsid and a first vector genome comprising a 5′ ITR, a U6 promoter, a sgRNA comprising at least 20 nucleotides that specifically bind to a target site in the PCSK9 gene, said target site being 5′ to a protospacer-adjacent motif (PAM) that is specifically recognized by the Cas9, a 5′ nuclear localization signal (NLS), a sequence encoding a Cas9 and regulatory sequences that direct expression of the Cas9 in a target cell comprising the PCSK9 gene, a 3′ NLS, and a 3′ ITR;
• and
• (b) a donor AAV vector comprising an AAV capsid and a second vector genome comprising:
• a 5′ITR, a 5′ homology directed recombination (HDR) arm, a transgene and regulatory sequences that direct expression of the transgene in the target cell, a 3′ HDR arm, and a 3′ ITR.

A system for treating a genetic disorder, the system comprising:

• (a) a gene editing vector comprising:
• (i) a lipid nanoparticle;
• (ii) a sgRNA comprising at least 20 nucleotides that specifically bind to a target site in the PCSK9 gene, said target site being 5′ to a protospacer-adjacent motif (PAM) that is specifically recognized by a Cas9;
• (iii) an mRNA comprising a 5′ nuclear localization signal (NLS), a sequence encoding the Cas9, a 3′ NLS;
• and
• (b) a donor AAV vector comprising an AAV capsid and a second vector genome comprising:
• a 5′ITR, a 5′ homology directed recombination (HDR) arm, a transgene and regulatory sequences that direct expression of the transgene in the target cell, a 3′ HDR arm, and a 3′ ITR.

A method of treating a liver metabolic disorder in a neonate subject, the method comprising:

• co-administering to the subject having a liver metabolic disorder:
• (a) a gene editing AAV vector comprising a sequence encoding a nuclease and regulatory sequences that direct expression of the nuclease in a target cell comprising a PCSK9 gene;
• and
• (b) a donor AAV vector comprising a transgene and regulatory sequences that direct expression of the transgene in the target cell, the donor vector further comprising homology-directed recombination (HDR) arms 5′ and 3′ to the transgene cassette.

A system for treating a genetic disorder, the system comprising:

• (a) a gene editing vector comprising a nucleic acid sequence encoding a nuclease;
• and
• (b) a donor vector comprising a nucleic acid sequence encoding an exogenous product for expression from the PCSK9 locus, wherein the inserted nucleic acid sequence does not encode PCSK9,
• wherein the system further comprises sequences that direct the nuclease to specifically targets the native PCSK9 gene locus;
• and
• wherein the native PCSK9 in the target cell is optionally ablated or reduced post-dosing with the dual vector system.