Abstract
Purpose:
To test for potential recombinations of human ND4 with mouse mtDNA.
Methods:
We injected into the mouse vitreous AAV to which the COX8 mitochondrial leader was appended to the VP2 capsid to deliver human ND4 to mitochondria. Controls consisted of AAV containing human ND4 but lacking the COX8 MTS or AAV-GFP. MtDNA was extracted from pooled infected retinas, then paired-end 100 base pair sequencing reads generated. Sequence alignment was performed with the Burroughs-Wheeler Aligner. 2x100bp paired-end reads from the Illumina Hiseq2000 were separately aligned to human and mouse genomes, as well as to human AAV-ND4. To identify potential mitochondrial insertions of the construct, we searched for paired-end reads, then for microscale homologous recombination events between human and mouse ND4.
Results:
(1) For MTS AAV-ND4, 579 read fragments aligned to human ND4. For AAV-ND4 lacking the MTS, only 10 read fragments were identified, none for AAV-GFP. (2) Examination of all putative chimeric read pairs at the 5’ end of human ND4 revealed only vector sequences not mouse ND4L that is immediately upstream of mouse ND4. At the 3’ prime end of human ND4 the attached FLAG epitope and vector sequences were detected, but not the tRNA histidine that is immediately downstream of mouse ND4. (3) Examination of mouse mitochondrial sequences for evidence of small-scale homologous recombination yielded no significant stretches attributable to human ND4 integration. While sporadic instances of single bases matched the human sequence, there were no stretches that could be distinguished from chance. (4) We found 43,079 100 base pair reads of mouse mtDNA with COX8 MTS-AAV-ND4, 45,505 reads with standard AAV-ND4 and 26,672 reads with AAV-GFP. Spanning the 16,000 bases of the mouse mitochondrial genome, in no instances did human ND4 insert into any site of mouse mtDNA. (5) As these findings indicated human ND4 remained episomal, we checked for mitochondrial depletion by preferential replication of the smaller human ND4 (1.4 Kb). Looking at the ratio of no of mitochondrial reads / total genomic reads - mitochondrial reads, ratios were 0.00973 (COX8 AAV-ND4), 0.01273 (AAV-ND4) and 0.00566 (AAV-GFP).
Conclusions:
The absence of (a) recombination events between mouse mtDNA and MTS transferred human ND4, (b) insertions into the host mtDNA or (c) mitochondrial depletion suggest that MTS-AAV gene transfer for treatment of LHON is safe.
Keywords: 613 neuro-ophthalmology: optic nerve •
600 mitochondria •
538 gene transfer/gene therapy