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Ornithine transcarbamylase deficiency (OTCD) is an X-linked liver disorder caused by partial or total loss of OTC enzyme activity. It is characterized by elevated plasma ammonia, leading to neurological impairments, coma, and death in the most severe cases. OTCD is managed by combining dietary restrictions, essential amino acids, and ammonia scavengers. However, to date, liver transplantation provides the best therapeutic outcome. AAV-mediated gene-replacement therapy represents a promising curative strategy. Here, we generated an AAV2/8 vector expressing a codon-optimized human OTC cDNA by the α1-AAT liver-specific promoter. Unlike standard codon-optimization approaches, we performed multiple codon-optimization rounds via common algorithms and ortholog sequence analysis that significantly improved mRNA translatability and therapeutic efficacy. AAV8-hOTC-CO (codon optimized) vector injection into adult OTCSpf-Ash mice (5.0E11 vg/kg) mediated long-term complete correction of the phenotype. Adeno-Associated viral (AAV) vector treatment restored the physiological ammonia detoxification liver function, as indicated by urinary orotic acid normalization and by conferring full protection against an ammonia challenge. Removal of liver-specific transcription factor binding sites from the AAV backbone did not affect gene expression levels, with a potential improvement in safety. These results demonstrate that AAV8-hOTC-CO gene transfer is safe and results in sustained correction of OTCD in mice, supporting the translation of this approach to the clinic., Graphical Abstract, This study presents an efficient AAV-mediated liver-specific gene therapy for OTCD, with improved vector safety features, resulting in steady hOTC liver expression and complete phenotype normalization of Spf-Ash mice, even after an ammonia challenge, at relatively low vector doses. These results support the potential translation of this strategy to the clinic.

Figure showing quantification of azelaic acid by HPLC on C18 reverse phase column. Quantification of azelaic acid (PSA produced) by standard curve A&B. (PPTX 51 kb)

Figure showing Quantification of azelaic acid by LC-MS. LC-MS profile of standard azelaic acid and putative azelaic acid collected at RT 13 from HPLC (A). Quantification of isolated and purified azelaic acid by standard curve (B). In 5 μl of RT 13 sample injected, there is 0.476 μg of azelaic acid which can be translated to 19.992 μg/L. (PPTX 69 kb)

Figure showing validation of the RNAseq-based expression by gene promoter studies. Promoters of selected gene were fused with promoter probe vector pBBRGFP-Gm and tested with similar concentration of azelaic acid which was used in RNAseq expression studies. Significant fold change from selected genes was observed only between pBGFPgene 1818/methanol and pBGFPgene 1818/azelaic acid. Data shown are the mean and standard deviation of experiment performed thrice in triplicate. (PPTX 99 kb)

Table representing RNAseq expressed genes upon induction of azeliac acid. (DOCX 15 kb)

Table showing list of compounds identified by NIST library search of PSV metabolome cultured in M9 glucose medium. (DOCX 21 kb)

Table showing list of compounds identified by NIST library search of PSA metabolome cultured in M9 glucose medium. (DOCX 24 kb)

Table showing list of compounds identified by NIST library search of M9 glucose medium Control. (DOCX 25 kb)

Both versions of C-terminal region of human nucleolin ChNu (646-706), containing Arg (I), respectively Nω ; , Nω ; -dimethylarginine (II), from the family of RGG-rich proteins, were successfully synthesized by an automated Fmoc methodology for SPPS using dimethoxybenzyl as a reversible amide bond protecting group. The cleavage/deprotection conditions were optimized and ChNu's were purified to homogeneity >95% by a combination of IE- and RP-HPLC. The purity/identity of peptides were confirmed by ESI-MS, AAA, SDS-PAGE, RP HPLC in two different systems and by IE HPLC. Arg-dimethylation increases hydrophobicity, as determined by RP-HPLC, and decreases basicity (IE-HPLC). In double filter binding assays (I) and (II) show very similar binding potency to single-stranded DNA (ssDNA) with values comparable to that of recombinant fusion ChNu (Kd ~ 2X10-7 M). The nucleic acid binding affinity is substantially increased in comparison to our published result on short peptides and correlates with the higher number of RGG, respectively aDma-GG-repeats. In a ssDNA-agarose affinity chromatography experiment Arg-dimethylation decreased the electrostatic interaction with ssDNA. Both (I) and (II) show very similar binding potency to G4 DNA and do not exhibit helicase activity in a M13 phage assay. (I) is efficiently, but not exhaustively methylated in vitro by recombinant protein arginine N-methyltransferase type I. 1H NMR spectra of (I) and (II) are characterized by the lack of dispersion for NH and Hα ; ; chemical shifts suggesting the absence of globular structure.