2020-02-15| Technology

New ‘Minigene’ Insertion Approach Could Treat Rare Liver Disease in Mice

by Ruchi Jhonsa
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By Ruchi Jhonsa, Ph.D.

Ornithine transcarbamylase (OTC) deficiency is an X linked, rare genetic disorder that is caused by a complete or partial lack of enzyme ornithine transcarbamylase. Resulting in an excessive accumulation of ammonia in the blood, OTC deficiency can lead to neurological abnormalities, reduced muscle tone and enlarged liver, respiratory abnormalities and eventually coma. Although the occurrence of OTC disorder is rare (1 in 40000), it can occur by genetic aberrations at more than 300 different regions on the gene. Current therapeutic methods aim at suppressing the symptoms by reducing protein amount in the food or supplementing citrulline or ornithine or essential amino acids in the diet. However, when ammonia levels shoot up in the blood, a drug intervention is needed to clear it. Buphenyl and Ravicti by Hyperion Therapeutics and Ammonul by Valeant Pharmaceuticals are the only drugs approved by the FDA for the treatment of chronic hyperammonemia. Though it is possible to dampen the symptoms, patients are still at high risk of mortality and effective therapies are needed to cure it.

Adeno-associated virus (AAV) based therapy that delivers the gene to the target cell could provide an alternative to current treatment options. However, because of the nonintegrating nature of AAV-mediated gene therapy, it is only effective for the short term as a vector genome will be lost during hepatocyte proliferation. Therefore, direct integration of the OTC transgene into the host genome will be the best method to treat the disease.

A new study from UPenn found a solution to this problem by looking down at the gene level. Researchers at UPenn devised a CRISPR based gene insertion tool that could potentially treat patients with OTC deficiency as well as other hereditary diseases triggered by different mutations on the same gene. The existing technology published in 2016 targets a single mutation and benefits only newborn mice, but this improved version can target any of the 300 known mutations on the OTC gene and provide long term effects that sustain in the adult mice.

But, how does this technology work? The team developed a dual AAV vector that specifically targets liver cells and contains two components- a Cas9 protein, and a ‘minigene’ expressing a codon-optimized human OTC. In the first step, the Cas9 enzyme delivered by the first AAV8 breaks the DNA at the target site. Immediately after, the second AAV delivers the minigene, which will be inserted in the genome by homology-directed repair (HDR). It was observed that mice treated with the targeted vector showed 25 and 35 percent of OTC-expressing cells in the liver at three and eight weeks, respectively, which is four and three-fold higher than the mice treated with untargeted vector. A significant reduction was also observed in ammonia levels in targeted mice compared to untreated mice fed on a protein-rich diet.

“Unlike other CRISPR approaches that delete or modify a portion of the normal gene, this technique integrates a new portion”, said lead author Lili Wang, Ph.D., a research associate professor of Medicine. We are not trying to correct mutation that stop liver cells from producing OTC, we are adding this new minigene so the cells can.” This property of the tool makes it broadly applicable. Like most monogenic diseases, OTCD is caused by more than 300 different mutations on the same gene. A CRISPR based correction method would require different guide RNA for correcting different mutations. But with the CRISPR based targeted insertion, a single guide RNA will be sufficient for all the different mutations.

“Like most genetic diseases that present lethal effects in newborns, early treatments that are effective for the long term are essential. Here, we moved a CRISPR approach forward to not only sustain the expression of OTC in the cells but also broaden the tool’s abilities. Our goal is to eventually translate this gene-editing approach from animals to treat patients with OTC disorders and other genetic diseases caused by mutations scattered throughout the gene rather than a single predominant mutation.” said James Wilson, MD, Ph.D., a professor of Medicine, and director of the Gene Therapy Program and the Orphan Disease Center at Penn.

Related Article: Using the Power of Multiplex Genome Engineering to Treat Cancer


  2. Wang et al., 2020, Sci. Adv. 6


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