AAV-CRISPR Persistence in the Eye of the Beholder
Authors: Alessandra Recchia
Journal: Molecular Therapy (2018)

Despite advances in genome editing technologies based on the adeno-associated virus (AAV)-CRISPR system, there are still concerns about the long-term persistence of recombinant AAV vectors in several organs (liver, muscle, eye) possibly leading to cytotoxicity or genotoxicity related to off-target effects. Indeed, there are still unanswered questions about long-lasting in vivo AAV persistence as a linear or circular DNA that is not targeted by epigenetic silencing in many tissues. In 2017, Kim et al.1Kim E. Koo T. Park S.W. Kim D. Kim K. Cho H.Y. Song D.W. Lee K.J. Jung M.H. Kim S. et al.In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni.Nat. Commun. 2017; 8: 14500Crossref PubMed Scopus (401) Google Scholar reported an editing approach based on AAV-CjCas9 to downregulate Vegfa or the hypoxia-inducible transcription factor Hif1a in mice displaying age-related macular degeneration (AMD)-related pathological choroidal neovascularization (CNV) induced by laser treatment. Although partial knockdown of either Vegfa or Hif1a provided benefits and reduced the area of CNV, local opsin dysfunction near the Vegfa-edited cells of murine retinal pigment epithelium (RPE) was observed. Conversely, no cone dysfunction was reported upon Hif1a partial knockdown. Lastly, no genome-wide off-target indels, evaluated 6 weeks after intravitreal injection of AAV-CjCas9 vector, were scored, indicating that prolonged expression of AAV-CjCas9 in vivo did not aggravate the genotoxic risk associated with the CjCas9 nuclease. In this issue of Molecular Therapy, the authors now report a long-term (14 months) safety study on C57BL/6J mice intravitreally injected with AAV-CjCas9 vectors targeting Vegfa or Hif1a genes.2Jo D.H. Koo T. Cho C.S. Kim J.H. Kim J.S. Kim J.H. Long-Term Effects of In Vivo Genome Editing in the Mouse Retina Using Campylobacter jejuni Cas9 Expressed via Adeno-Associated Virus.Mol Ther. 2018; 27: 130-136Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar The findings continue to show that the AAV-CRISPR system in the eyes is long lasting, effective, and safe. CRISPR/Cas9 genome editing in the retina represents a potential treatment strategy for inherited retinal dystrophies (e.g., autosomal dominant retinitis pigmentosa [adRP] and Leber congenital amaurosis [LCA]) and retinal neovascular diseases (e.g., wet AMD and proliferative diabetic retinopathy). CRISPR components have been delivered to the retina by viral and non-viral methods. Although subretinal plasmid electroporation is not suitable for therapeutic interventions in patients, it has been employed to knock down a mutant Rhodopsin gene in mouse3Latella M.C. Di Salvo M.T. Cocchiarella F. Benati D. Grisendi G. Comitato A. Marigo V. Recchia A. In vivo Editing of the Human Mutant Rhodopsin Gene by Electroporation of Plasmid-based CRISPR/Cas9 in the Mouse Retina.Mol. Ther. Nucleic Acids. 2016; 5: e389Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar and rat4Bakondi B. Lv W. Lu B. Jones M.K. Tsai Y. Kim K.J. Levy R. Akhtar A.A. Breunig J.J. Svendsen C.N. Wang S. In Vivo CRISPR/Cas9 Gene Editing Corrects Retinal Dystrophy in the S334ter-3 Rat Model of Autosomal Dominant Retinitis Pigmentosa.Mol Ther. 2015; 24: 556-563Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar models of adRP. Recently, preassembled Vegfa-specific Cas9 ribonucleoproteins (RNPs) have been subretinally injected into a mouse model of AMD, demonstrating a significant reduction of laser-induced CNV. However, the effects were localized only to the injected area of RPE, with no transduction of the neural retina.5Kim K. Park S.W. Kim J.H. Lee S.H. Kim D. Koo T. Kim K.E. Kim J.H. Kim J.S. Genome surgery using Cas9 ribonucleoproteins for the treatment of age-related macular degeneration.Genome Res. 2017; 27: 419-426Crossref PubMed Scopus (112) Google Scholar Nonvirally-mediated transient expression of CRISPR components in the retina may reduce safety concerns, although viral delivery systems based on AAV represent the most efficient and safe tools for gene delivery to the retina. Indeed genome editing using the AAV-CRISPR system has been widely reported as efficient, safe, and precise in more than 30 published studies in mouse models6Lau C.H. Suh Y. In vivo genome editing in animals using AAV-CRISPR system: applications to translational research of human disease.F1000Res. 2017; 6: 2153Crossref PubMed Scopus (98) Google Scholar of diseases associated with the eyes, muscle, liver, heart, and lung. Despite the great potential of AAV vectors, their relatively small packaging capacity represents a limitation for delivering the widely used Streptococcus pyogenes Cas9 (SpCas9) together with guide RNAs (gRNAs) and large transgenes. Dual-vector AAV systems, smaller Cas9 orthologs, or other nucleases belonging to the type-V CRISPR-Cas systems (AsCpf1 and LbCpf1) have circumvented the transgene packaging issue. Packaging of SpCas9 and gRNA into two separate AAV particles proved to be a successful delivery strategy.7Giannelli S.G. Luoni M. Castoldi V. Massimino L. Cabassi T. Angeloni D. Demontis G.C. Leocani L. Andreazzoli M. Broccoli V. Cas9/sgRNA selective targeting of the P23H Rhodopsin mutant allele for treating retinitis pigmentosa by intravitreal AAV9.PHP.B-based delivery.Hum. Mol. Genet. 2018; 27: 761-779Crossref PubMed Scopus (90) Google Scholar Similarly, an AAV-split-Cas9 system has been developed. These strategies rely on the possibility to reassemble the Cas9 holoenzyme in transduced cells by fusing the N- and C-terminal domains of Cas9 to Rapamycin-inducible FRB-FKBP dimerization domains8Zetsche B. Volz S.E. Zhang F. A split-Cas9 architecture for inducible genome editing and transcription modulation.Nat. Biotechnol. 2015; 33: 139-142Crossref PubMed Scopus (472) Google Scholar or Intein, thus triggering trans-splicing of Intein-tagged Cas9 domains.9Chew W.L. Tabebordbar M. Cheng J.K. Mali P. Wu E.Y. Ng A.H. Zhu K. Wagers A.J. Church G.M. A multifunctional AAV-CRISPR-Cas9 and its host response.Nat. Methods. 2016; 13: 868-874Crossref PubMed Scopus (373) Google Scholar Smaller Cas9 orthologs from Staphylococcus aureus (SaCas9),10Ran F.A. Cong L. Yan W.X. Scott D.A. Gootenberg J.S. Kriz A.J. Zetsche B. Shalem O. Wu X. Makarova K.S. et al.In vivo genome editing using Staphylococcus aureus Cas9.Nature. 2015; 520: 186-191Crossref PubMed Scopus (1769) Google Scholar Campylobacter jejuni (CjCas9),1Kim E. Koo T. Park S.W. Kim D. Kim K. Cho H.Y. Song D.W. Lee K.J. Jung M.H. Kim S. et al.In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni.Nat. Commun. 2017; 8: 14500Crossref PubMed Scopus (401) Google Scholar Streptococcus thermophilus (StCas9),11Kleinstiver B.P. Prew M.S. Tsai S.Q. Topkar V.V. Nguyen N.T. Zheng Z. Gonzales A.P. Li Z. Peterson R.T. Yeh J.R. et al.Engineered CRISPR-Cas9 nucleases with altered PAM specificities.Nature. 2015; 523: 481-485Crossref PubMed Scopus (1069) Google Scholar and Neisseria meningitidis (NmCas9)12Esvelt K.M. Mali P. Braff J.L. Moosburner M. Yaung S.J. Church G.M. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing.Nat. Methods. 2013; 10: 1116-1121Crossref PubMed Scopus (604) Google Scholar have also been isolated. Owing to their smaller size (2.9–3.3 kb) compared to SpCas9, they potentially could be suitable for packaging into a single AAV vector also bearing the gRNA. However, StCas9 and NmCas9 have been neglected owing to their longer protospacer adjacent motif (PAM), which represents a limitation on sequences available for targeting. Conversely, SaCas9 and CjCas9 have been successfully vectorized together with gRNAs in a single AAV and show cutting ability comparable to SpCas9 in in vitro applications.5Kim K. Park S.W. Kim J.H. Lee S.H. Kim D. Koo T. Kim K.E. Kim J.H. Kim J.S. Genome surgery using Cas9 ribonucleoproteins for the treatment of age-related macular degeneration.Genome Res. 2017; 27: 419-426Crossref PubMed Scopus (112) Google Scholar More recently, Cpf1 nuclease isolated from Lachnospiraceae bacterium (LbCpf1) has been vectorized together with the cognate CRISPR RNA (crRNA) into a single AAV vector,13Koo T. Park S.W. Jo D.H. Kim D. Kim J.H. Cho H.Y. Kim J. Kim J.H. Kim J.S. CRISPR-LbCpf1 prevents choroidal neovascularization in a mouse model of age-related macular degeneration.Nat. Commun. 2018; 9: 1855Crossref PubMed Scopus (56) Google Scholar demonstrating its great potential as an in vivo genome editing tool for the treatment of angiogenesis-related diseases. AAV-CRISPR systems based on CjCas9 and LbCpcf1 nucleases have been developed to treat AMD, and AAV-CRISPR persistence and safety issues have been examined in mice.14Hakim C.H. Wasala N.B. Nelson C.E. Wasala L.P. Yue Y. Louderman J.A. Lessa T.B. Dai A. Zhang K. Jenkins G.J. et al.AAV CRISPR editing rescues cardiac and muscle function for 18 months in dystrophic mice.JCI Insight. 2018; 3: e124297Crossref PubMed Scopus (64) Google Scholar, 15Villiger L. Grisch-Chan H.M. Lindsay H. Ringnalda F. Pogliano C.B. Allegri G. Fingerhut R. Häberle J. Matos J. Robinson M.D. et al.Treatment of a metabolic liver disease by in vivo genome base editing in adult mice.Nat Med. 2018; 24: 1519-1525Crossref PubMed Scopus (211) Google Scholar Currently, patients with wet AMD benefit from intravitreal injection of anti-vascular endothelial growth factor (VEGF) agents (e.g., ranibizumab, bevacizumab, and aflibercept). However, there is a compelling need for a long-lasting therapy solution for patients with AMD because of the cost of the drugs, patient access to treatment, and the increasing number of patients as the population ages. In the new study, the authors showed no changes in histologic integrity and function of retinal tissues treated with AAV-CjCas9.2Jo D.H. Koo T. Cho C.S. Kim J.H. Kim J.S. Kim J.H. Long-Term Effects of In Vivo Genome Editing in the Mouse Retina Using Campylobacter jejuni Cas9 Expressed via Adeno-Associated Virus.Mol Ther. 2018; 27: 130-136Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar Then they reported an efficient editing in both target genes 6 weeks post-injection, which clearly increased 8 weeks later in both retina and RPE, although Vegfa-specific indels arose only in the retina. These data indicate the long-term persistence of constitutively expressed and active CjCas9 nuclease in the injected area. Lastly, the authors analyzed the potential off-target sites by targeted deep sequencing and performed a comprehensive genome-wide analysis of potential off-targets sites bearing up to 4 nucleotide mismatches in the mouse genome. Any detectable indels were scored in 21 homologous sites. Altogether, this study demonstrated that intravitreal injection of AAV-CjCas9 vector targeted to Hif1a effectively induced and maintained mutations in murine retinal cells for 14 months without giving rise to off-target indels due to the constitutively active CjCas9 nuclease and without affecting retinal histologic integrity or function. Unfortunately, no data on CNV amelioration 14 months post-injection are reported. It would have been extremely useful to see whether higher indel frequency at target sites resulted in more effective treatment of wet AMD. Other AAV-CRISPR systems developed for treatment of adRP or LCA 107Giannelli S.G. Luoni M. Castoldi V. Massimino L. Cabassi T. Angeloni D. Demontis G.C. Leocani L. Andreazzoli M. Broccoli V. Cas9/sgRNA selective targeting of the P23H Rhodopsin mutant allele for treating retinitis pigmentosa by intravitreal AAV9.PHP.B-based delivery.Hum. Mol. Genet. 2018; 27: 761-779Crossref PubMed Scopus (90) Google Scholar, 16Tsai Y.T. Wu W.H. Lee T.T. Wu W.P. Xu C.L. Park K.S. Cui X. Justus S. Lin C.S. Jauregui R. et al.Clustered Regularly Interspaced Short Palindromic Repeats-Based Genome Surgery for the Treatment of Autosomal Dominant Retinitis Pigmentosa.Ophthalmology. 2018; 125: 1421-1430Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 17Yu W. Mookherjee S. Chaitankar V. Hiriyanna S. Kim J.W. Brooks M. Ataeijannati Y. Sun X. Dong L. Li T. et al.Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice.Nat. Commun. 2017; 8: 14716Crossref PubMed Scopus (196) Google Scholar, 18Ruan G.X. Barry E. Yu D. Lukason M. Cheng S.H. Scaria A. CRISPR/Cas9-Mediated Genome Editing as a Therapeutic Approach for Leber Congenital Amaurosis 10.Mol. Ther. 2017; 25: 331-341Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar showed prolonged and safe expression (up to 9.5 months) of subretinal-injected AAV-SpCas9 targeting specific mutations in RHO or CEP290 genes or targeting neural retina leucine zipper transcription factor (Nrl) in mouse models for adRP. Although, in these studies, the authors employed different AAV serotypes and different vector doses and targeted different genes, the results showed effective rescue of RP or LCA 10 phenotype without toxicity or off-target effects. We now have a potent molecular tool to employ in several eye disorders because Kim's group demonstrated a robust in vivo knock down of a murine transcription factor, perfectly conserved in the human genome and involved in tumor angiogenesis and pathophysiology of ischemic disease, and maybe this strategy could be translated to other vascularization-associated human diseases. The author gratefully acknowledges support from Fondazione Roma, Call Retinitis Pigmentosa. Long-Term Effects of In Vivo Genome Editing in the Mouse Retina Using Campylobacter jejuni Cas9 Expressed via Adeno-Associated VirusJo et al.Molecular TherapyOctober 16, 2018In BriefJo et al. demonstrated that intravitreally administered adeno-associated virus encoding the Cas9 ortholog from Campylobacter jejuni targeted to Hif1a did not induce histologic and functional aberration in the retina. Despite the accumulation of on-target insertions and deletions (indels), there were no indels at the potential off-target sites. Full-Text PDF Open Archive