Late-Onset Ornithine Transcarbamylase Deficiency Complicated with Extremely High Serum Ammonia Level: Prompt Induction of Hemodialysis as the Key to Successful Treatment.

BACKGROUND Ornithine transcarbamylase deficiency (OTCD) is an X-linked semi-dominant disorder, causing possible fatal hyperammonemia. Late-onset OTCD can develop at any time from 2 months after birth to adulthood, accounting for 70% of all OTCDs. CASE REPORT A 35-year-old man with chronic headaches stated that since childhood he felt sick after eating meat. Fourteen days before hospital admission, he began receiving 60 mg/day of intravenous prednisolone for sudden deafness. The prednisolone was stopped 5 days before hospital admission. Four days later, he was transferred to our hospital because of confusion. On admission, he had hyperammonemia of 393 µmol/L. Because he became comatose 7 hours after admission, and his serum ammonia increased to 1071 µmol/L, we promptly started hemodialysis. Because his family history included 2 deceased infant boys, we suspected late-onset OTCD. On day 2 of hospitalization, we began administering ammonia-scavenging medications. Because he gradually regained consciousness, we stopped his hemodialysis on day 6. After his general condition improved, he was transferred to the previous hospital for rehabilitation on day 32. We definitively diagnosed him with late-onset OTCD due to the low plasma citrulline and high urinary orotic acid levels found during his hospitalization. CONCLUSIONS Clinicians should suspect urea cycle disorders, such as OTCD, when adult patients present with marked hyperammonemia without liver cirrhosis. Adult patients with marked hyperammonemia should immediately undergo hemodialysis to remove ammonia, regardless of causative diseases.

Effects of Self-Complementarity, Codon Optimization, Transgene, and Dose on Liver Transduction with AAV8.

Numerous methods of vector design and delivery have been employed in an attempt to increase transgene expression following AAV-based gene therapy. Here, a gene transfer study was conducted in mice to compare the effects of vector self-complementarity (double- or single-stranded DNA), codon optimization of the transgene, and vector dose on transgene expression levels in the liver. Two different reporter genes were used: human ornithine transcarbamylase (hOTC) detected by immunofluorescence, and enhanced green fluorescent protein (EGFP) detected by direct fluorescence. The AAV8 capsid was chosen for all experiments due to its strong liver tropism. While EGFP is already a codon-optimized version of the original gene, both wild-type (WT) and codon-optimized (co) versions of the hOTC transgene were compared in this study. In addition, the study evaluated which of the two hOTC modifications-codon optimization or self-complementarity-would confer the highest increase in expression levels at a given dose. Interestingly, based on morphometric image analysis, it was observed that the difference in detectable expression levels between self-complementary (sc) and single-stranded (ss) hOTCco vectors was dose dependent, with a sevenfold increase in OTC-positive area using sc vectors at a dose of 3 × 109 genome copies (GC) per mouse, but no significant difference at a dose of 1 × 1010 GC/mouse. In contrast, with EGFP as a transgene, the increases in expression levels when using the sc vector were observed at both the 3 × 109 GC/mouse and 1 × 1010 GC/mouse doses. Furthermore, codon optimization of the hOTC transgene generated a more significant improvement in expression than the use of self-complementarity did. Overall, the results demonstrate that increases in expression levels gained by using sc vectors instead of ss vectors can vary between different transgenes, and that codon optimization of the transgene can have an even more powerful effect on the resulting expression levels.

A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice.

Many genetic liver diseases in newborns cause repeated, often lethal, metabolic crises. Gene therapy using nonintegrating viruses such as adeno-associated virus (AAV) is not optimal in this setting because the nonintegrating genome is lost as developing hepatocytes proliferate. We reasoned that newborn liver may be an ideal setting for AAV-mediated gene correction using CRISPR-Cas9. Here we intravenously infuse two AAVs, one expressing Cas9 and the other expressing a guide RNA and the donor DNA, into newborn mice with a partial deficiency in the urea cycle disorder enzyme, ornithine transcarbamylase (OTC). This resulted in reversion of the mutation in 10% (6.7-20.1%) of hepatocytes and increased survival in mice challenged with a high-protein diet, which exacerbates disease. Gene correction in adult OTC-deficient mice was lower and accompanied by larger deletions that ablated residual expression from the endogenous OTC gene, leading to diminished protein tolerance and lethal hyperammonemia on a chow diet.

Recombinant adeno-associated virus integration sites in murine liver after ornithine transcarbamylase gene correction.

Recombinant adeno-associated viruses (rAAVs) have been tested in humans and other large mammals without adverse events. However, one study of mucopolysaccharidosis VII correction in mice showed repeated integration of rAAV in cells from hepatocellular carcinoma (HCC) in the Dlk1-Dio3 locus, suggesting possible insertional mutagenesis. In contrast, another study found no association of rAAV integration with HCC, raising questions about the generality of associations between liver transformation and integration at Dlk1-Dio3. Here we report that in rAAV-treated ornithine transcarbamylase (Otc)-deficient mice, four examples of integration sites in Dlk1-Dio3 could be detected in specimens from liver nodule/tumors, confirming previous studies of rAAV integration in the Dlk1-Dio3 locus in the setting of another murine model of metabolic disease. In one case, the integrated vector was verified to be present at about one copy per cell, consistent with clonal expansion. Another verified integration site in liver nodule/tumor tissue near the Tax1bp1 gene was also detected at about one copy per cell. The Dlk1-Dio3 region has also been implicated in human HCC and so warrants careful monitoring in ongoing human clinical trials with rAAV vectors.

Biotechnology and clinical trials.

Not surprisingly, clinical trials have been critically important to developments in the field of biotechnology. Perhaps less expectedly, the clinical trials of biotechnology products have been critically important to recent developments in the field of clinical trials design, conduct, and analysis. This manuscript explores three examples of biotechnology clinical trials--a trial in sepsis, a trial in fibrinolytics in myocardial infarction, and trials in gene therapy--and highlights their contributions to the theory and practice of clinical research.