May 1, 2017 (Vol. 37, No. 9)

Terence R. Flotte M.D. Editor-in-Chief Human Gene Therapy, Mary Ann Lieber, Inc.

Genome Editing Is Particularly Appropriate for This Disorder

α1-Antitrypsin deficiency (AATD) is a single-gene disorder, particularly common in Scandinavia, Ireland, Northern Europe, and North America. The carrier frequency, which is approximately 3% in the United States, reaches as high as 25% among chronic lung disease patients in Ireland, where a very strong founder effect may account for the high incidence of one particular missense mutation, designated as protease inhibitor type Z (PiZ, E342K). AATD is peculiar among genetic diseases in that the common mutation can lead to two distinct pathologies.

AAT is primarily produced in hepatocytes, and PiZ-AAT polymerizes in the endoplasmic reticulum, resulting in impaired secretion. Most commonly, this simply causes a serum deficiency of anti-elastase function, which leads to damage of the pulmonary interstitium and subsequent chronic lung disease. In a subset of PiZ homozygotes, this polymerization triggers liver disease as well, because of damaging effects of mutant protein aggregates in hepatocytes.

Interestingly, the burden of mutant PiZ-AAT in hepatocytes may make AATD patients prone to enhanced liver toxicity from viral vectors, which has led to a variety of approaches to gene augmentation of the wild-type (PiM) AAT, intended to at least be “liver-sparing” if not “liver-treating.”

The investigative team at the Horae Gene Therapy Center at the University of Massachusetts Medical School is approaching this problem in a number of distinct ways. Among the most promising is correction of the common PiZ mutant allele by CRISPR-mediated genome editing.

Genome editing is particularly appropriate for this disorder. Insertions or deletions (indels) could be introduced within the PiZ locus by creating double-strand breaks (DSBs) using Cas9 and an appropriate sgRNA, thereby triggering non-homologous end joining (NHEJ). This could be beneficial for prevention of AATD liver disease, if combined with some method of protein or gene augmentation for PiM.

More definitively, AATD due to the PiZ mutation could be corrected by homology-directed repair (HDR), in which a DSB is introduced near the site of the mutation and combined with a single-stranded DNA template spanning the mutation site, so that the DNA repair machinery results in the generation of a wild-type PiM allele sequence.

In order to accomplish NHEJ in vivo within hepatocytes, a number of gene transfer strategies may be used to introduce both the sgRNA and the editing enzyme. For in vivo expression of Cas9 within a mouse liver, several methods could be used, including nonviral mediated DNA or RNA gene transfer or rAAV-mediated expression, which could be most easily done with Staphylococcus aureus Cas9 (SaCas9). The introduction of the single-stranded DNA template for HDR could also be accomplished in a number of different ways, with rAAV showing particular promise. As these technologies are developed, the clinical approaches and endpoints that have been used for nonediting-based gene therapy could be paralleled, allowing for clear and consistent endpoints for product licensure, as well as direct comparison of the various approaches.

Terence R. Flotte, M.D., is the dean of the School of Medicine, the provost and executive deputy chancellor, and the Celia and Isaac Haidak Professor of Medical Education at the University of Massachusetts Medical School.

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