325. In Vitro Gene Knockdown of Rat Pyruvate Dehydrogenase E1 α mRNA by Self- Complementary Adeno-Associated Virus-Mediated Small Interfering RNA Delivery

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Mutations in the E1 α subunit (PDHA1) of the gene pyruvate dehydrogenase complex (PDC) are one of the most common causes of congenital lactic acidosis. An animal model of the E1 α deficiency would provide insight into the pathologic role of PDC mutations in genetic mitochondrial diseases. Small interfering RNAs (siRNAs) designed to cleave the messenger RNA (mRNA) of the E1 α subunit, were constructed and tested in vitro to assess the feasibility of producing a gene knockdown in rats. HEK 293 cells were co- transfected with a rat PDHA1 expression vector and eight naked siRNAs that specifically targeted rat E1 α mRNA. Real time PCR (qPCR) analyses showed that four siRNAs reduced rat PDHA1 RNA levels up to 85% by 24 hours, and up to 65% by 56 hours, compared to negative and positive controls. Since oligonucleotide- mediated siRNA delivery provides only transient suppression, we next selected two siRNA candidates and generated self- complementary, double-stranded adeno-associated virus (scAAV) vectors (serotype 2 and 5) expressing a rat short hairpin siRNA expression cassette (scAAVsi-PDHA1). Rat lung fibroblast (RLF) cultures were infected with scAAVsi-PDHA1 vectors. The RLF PDHA1 mRNA level was reduced 53-80% 72 hours post-infection and 54-70% 10 days post-infection in RLF cultures. The expression of E1 α and the specific activity of pyruvate dehydrogenase were also decreased at 10 days post-infection in RLF cultures. Thus, AAV scAAV siRNA-mediated gene knockdown of PDHA1 provides strategy that may be applicable for creating a useful animal model of PDC deficiency. Mutations in the E1 α subunit (PDHA1) of the gene pyruvate dehydrogenase complex (PDC) are one of the most common causes of congenital lactic acidosis. An animal model of the E1 α deficiency would provide insight into the pathologic role of PDC mutations in genetic mitochondrial diseases. Small interfering RNAs (siRNAs) designed to cleave the messenger RNA (mRNA) of the E1 α subunit, were constructed and tested in vitro to assess the feasibility of producing a gene knockdown in rats. HEK 293 cells were co- transfected with a rat PDHA1 expression vector and eight naked siRNAs that specifically targeted rat E1 α mRNA. Real time PCR (qPCR) analyses showed that four siRNAs reduced rat PDHA1 RNA levels up to 85% by 24 hours, and up to 65% by 56 hours, compared to negative and positive controls. Since oligonucleotide- mediated siRNA delivery provides only transient suppression, we next selected two siRNA candidates and generated self- complementary, double-stranded adeno-associated virus (scAAV) vectors (serotype 2 and 5) expressing a rat short hairpin siRNA expression cassette (scAAVsi-PDHA1). Rat lung fibroblast (RLF) cultures were infected with scAAVsi-PDHA1 vectors. The RLF PDHA1 mRNA level was reduced 53-80% 72 hours post-infection and 54-70% 10 days post-infection in RLF cultures. The expression of E1 α and the specific activity of pyruvate dehydrogenase were also decreased at 10 days post-infection in RLF cultures. Thus, AAV scAAV siRNA-mediated gene knockdown of PDHA1 provides strategy that may be applicable for creating a useful animal model of PDC deficiency.