2 April 2019

Paper of the Month April 2019

Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy

Human genome has estimated 20,000-25,000 protein-coding genes. Over 3000 genes within these protein-coding genes are closely linked to genetic diseases. Advances in therapeutic genome editing enable correction of these mutations for many genetic diseases. Scientists have been using many different genome editing strategies including transcription activator-like effector nucleases (TALEN) and zinc-finger nuclease (ZFN) to introduce intended modifications to the host genome. The discovery of clustered regularly interspaced short palindromic repeats (CRISPR) technique has remarkably accelerated the development of gene editing in many different areas aiming for an improved targeting and efficiency with ease of use.

Duchenne muscular dystrophy (DMD) is a rare genetic disease caused by a mutation on DMD gene that codes for dystrophin and results in muscle weakness and degeneration. There is no established treatment for Duchenne muscular dystrophy which motivates scientists to develop new technique using the potential of CRISPR. There are many different approaches to introduce a transgene to the host genome. One of the most popular transgene delivery method is to use viral vectors including adenovirus, adeno-associated virus or lentivirus for correction of several kinds of genetic diseases. In this study, they have used AAV to make their intended modifications.

On this study, Nelson et al. has shown that the potential of AAV-CRISPR for permanent genome corrections and the most remarkable experiment they have done is that in addition to short-term effect of gene correction on mouse disease model they have also analyzed the sustainability of genome editing 1 year after a single intravenous administration of an adeno-associated virus that encodes CRISPR (AAV-CRISPR). Besides, they also examined that the immunogenicity and toxicity caused by AAV-CRISPR in the host cell and they analyzed humoral and cellular response.

To do so, they treated adult mice locally by intramuscular (i.m.) injection of AAV8 into the tibialis anterior and analyzed the results at two different time points: 8-weeks and 6-months after AAV-CRISPR administration. Secondly, they treated neonatal mice by AAV8 systemic injection via facial vein injection (FVI) which was followed by analysis at 8-weeks and 1-year. And finally, 8-weeks old mice have been examined with systemic injection of AAV and checked at two different time points: 8-weeks and 1-year. To analyze gene editing efficiency, they developed an Nextera-based unbiased deep-sequencing method in addition to droplet digital PCR (ddPCR) and PCR-based methods for deep sequencing due to its advantages.

They demonstrated that adult mice treated local AAV administration via i.m. injection had a significant decrease in genome-editing levels over time. However, when they treated systemically, genome-editing level has been increased over year. Besides unbiased deep-sequencing, they have also analyzed Dmd mRNA transcripts using ddPCR. These results have also showed the same profile with genome-editing level. As a functional assay, they verified restoration of dystrophin protein by immunofluorescence staining and western blot of cardiac and skeletal muscle. They clearly demonstrated that one year after AAV-CRISPR injection, the level of dystrophin expression have remained the same.

One of the notable results from the paper is a term “genotoxicity” of AAV-CRISPR complex. Based on deep sequencing of the top ten predicted off-target sites did not show a significant off-target increase, but they noted that AAV have been integrated to the host genome despite its episomal character. As it is known, AAV integration to the host genome is not a common case, but when the double strand break (DSB) is introduced to the host genome, AAV can also be inserted randomly which is definitely unwanted event for therapeutic genome editing. Besides the genotoxicity caused by AAV integration to the host genome, they have also detected a humoral and cellular immune response against the SaCas9 protein in 31 out of 32 mice. However, when neonatal mice treated, no humoral or cellular response has been detected regardless of administration route.

As a summary, they highlighted again that CRISPR has a remarkable potential for therapeutic use, however, it still needs to be improved in terms of cellular and/or humoral response and efficiency of genome editing. As they also shown on the paper, AAV-CRISPR was well-tolerated over a year without toxicity, but it might be integrated into the host genome causing genotoxicity. Thus, it is highly important to conduct wide range of pre-clinical experiments before moving further to therapeutic purposes and to characterize its safety and efficiency in different models.