Cas9-specific immune responses compromise local and systemic AAV CRISPR therapy in multiple dystrophic canine models

Abstract

Adeno-associated virus (AAV)-mediated CRISPR-Cas9 editing holds promise to treat many diseases. The immune response to bacterial-derived Cas9 has been speculated as a hurdle for AAV-CRISPR therapy. However, immunological consequences of AAV-mediated Cas9 expression have thus far not been thoroughly investigated in large mammals. We evaluate Cas9-specific immune responses in canine models of Duchenne muscular dystrophy (DMD) following intramuscular and intravenous AAV-CRISPR therapy. Treatment results initially in robust dystrophin restoration in affected dogs but also induces muscle inflammation, and Cas9-specific humoral and cytotoxic T-lymphocyte (CTL) responses that are not prevented by the muscle-specific promoter and transient prednisolone immune suppression. In normal dogs, AAV-mediated Cas9 expression induces similar, though milder, immune responses. In contrast, other therapeutic (micro-dystrophin and SERCA2a) and reporter (alkaline phosphatase, AP) vectors result in persistent expression without inducing muscle inflammation. Our results suggest Cas9 immunity may represent a critical barrier for AAV-CRISPR therapy in large mammals.

A universal strategy for AAV delivery of base editors to correct genetic point mutations in neonatal PKU mice

“… 35 Nevertheless, further research needs to be done to determine if the AAV-infected cells are removed by preexisting Cas9-specific immune responses, since the most widely used Cas9s, saCas9 and spCas9, are derived from common pathogens. 36 , 37 , 38 , 39 In addition, long-term investigations of AAV mediated base editing therapy are also required to evaluate the potential side effect of base editors, such as genome wide DNA and RNA off-target editing. 40 , 41 , 42 , 43 …”

Extensor carpi ulnaris muscle shows unexpected slow-to-fast fiber-type switch in Duchenne muscular dystrophy dogs

“…All experimental dogs were on a mixed genetic background of the golden retriever, Labrador retriever, beagle and Welsh corgi, and were generated in-house by artificial insemination. The genotype of the affected dogs was determined by PCR ( Fine et al, 2011 ; Hakim et al, 2021 ; Smith et al, 2011 ). All experimental dogs were housed in an American Association for Accreditation of Laboratory Animal Care-accredited, limited access, conventional animal care facility and kept under a 12-h light/12-h dark cycle.…”

CAR-T Regulatory (CAR-Treg) Cells: Engineering and Applications

“…Gene editing has the potential to revolutionize the gene therapy field, and with the discovery of the clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 system, the interest in the ability to precisely edit specific genes of interest has been enhanced [ 168 ]. However, studies have detected Cas9-associated humoral and cellular immune responses [ 169 , 170 , 171 ] and the prevalence of anti-Cas9 antibodies and T cells within the human population [ 172 , 173 ]. Although much success of CRISPR editing has been described for ex vivo therapies, numerous in vivo editing strategies have suggested immune responses to Cas9 may inhibit therapeutic effects [ 171 , 174 ].…”

“…However, studies have detected Cas9-associated humoral and cellular immune responses [ 169 , 170 , 171 ] and the prevalence of anti-Cas9 antibodies and T cells within the human population [ 172 , 173 ]. Although much success of CRISPR editing has been described for ex vivo therapies, numerous in vivo editing strategies have suggested immune responses to Cas9 may inhibit therapeutic effects [ 171 , 174 ]. Ferdosi et al modified Cas9 protein to eliminate immunodominant epitopes by utilizing targeted mutations while still preserving its function and specificity [ 175 ].…”