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Cited by 230CRISPR and Genetic EngineeringVirus-based gene therapy researchRNA Interference and Gene Delivery

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AAV-CRISPR Gene Editing Is Negated by Pre-existing Immunity to Cas9

Open access
Li, Ang, Tanner, Mark R., Lee, Ciaran M., Hurley, Ayrea E., De Giorgi, Marco, Jarrett, Kelsey E., Davis, Timothy H., Doerfler, Alexandria M., Bao, Gang, Beeton, Christine, Lagor, William R.·2020·Molecular Therapy
DOI 10.1016/j.ymthe.2020.04.017PMC PMC7264438
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Abstract

Adeno-associated viral (AAV) vectors are a leading candidate for the delivery of CRISPR-Cas9 for therapeutic genome editing in vivo . However, AAV-based delivery involves persistent expression of the Cas9 nuclease, a bacterial protein. Recent studies indicate a high prevalence of neutralizing antibodies and T cells specific to the commonly used Cas9 orthologs from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9) in humans. We tested in a mouse model whether pre-existing immunity to SaCas9 would pose a barrier to liver genome editing with AAV packaging CRISPR-Cas9. Although efficient genome editing occurred in mouse liver with pre-existing SaCas9 immunity, this was accompanied by an increased proportion of CD8 + T cells in the liver. This cytotoxic T cell response was characterized by hepatocyte apoptosis, loss of recombinant AAV genomes, and complete elimination of genome-edited cells, and was followed by compensatory liver regeneration. Our results raise important efficacy and safety concerns for CRISPR-Cas9-based in vivo genome editing in the liver.

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Crossref · 10.1016/j.ymthe.2020.04.017

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Graphical abstract CC BY-NC-ND · PubMed Central
Figure 1 Induction of Immune Memory against SaCas9 in C57BL/6 Mice (A) Mice were initially immunized with either purified ovalbumin (Ova) or SaCas9 protein. One week after immunization, mice were challenged with either ovalbumin or SaCas9, and ear thickness was measured after 24 h. (B) Histological analysis of the left ear of mice in (A) stained with hematoxylin and eosin. ∗p < 0.05, ∗∗p < 0.01; ns, not significant. Scale bar, 50 μm. (C) Representative H&E staining of mouse ears.
Figure 1 Induction of Immune Memory against SaCas9 in C57BL/6 Mice (A) Mice were initially immunized with either purified ovalbumin (Ova) or SaCas9 protein. One week after immunization, mice were challenged with either ovalbumin or SaCas9, and ear thickness was measured after 24 h. (B) Histological analysis of the left ear of mice in (A) stained with hematoxylin and eosin. ∗p < 0.05, ∗∗p < 0.01; ns, not significant. Scale bar, 50 μm. (C) Representative H&E staining of mouse ears.CC BY-NC-ND · PubMed Central
Figure 2 AAV-CRISPR Gene Editing of Mice Immunized against SaCas9 Elicits a Robust CD8+ T Cell Response (A) Mice were immunized with either ovalbumin or SaCas9 protein 1 week prior to co-delivery of AAV vectors encoding a gRNA targeting Ldlr with SaCas9 and GFP. Tissues were harvested at 1, 2, 4, 6, and 12 weeks post-injection. (B) CD8+ T cells as a percentage of total lymphocytes in the liver were measured by flow cytometry. (C) CD8 mRNA expression between mice immunized against ovalbumin and SaCas9 measured by qPCR. (D) Representative TUNEL staining in mouse liver sections. (E) Serum alanine transaminase (ALT) activity levels indicating liver damage. (F) Representative Ki-67 staining of mouse liver sections. (G) Ki-67 mRNA expression in mouse liver. Group sizes in (C), (E), and (G) are n = 7, except for ovalbumin 6 weeks (n = 6) and SaCas9 1 week (n = 6).
Figure 2 AAV-CRISPR Gene Editing of Mice Immunized against SaCas9 Elicits a Robust CD8+ T Cell Response (A) Mice were immunized with either ovalbumin or SaCas9 protein 1 week prior to co-delivery of AAV vectors encoding a gRNA targeting Ldlr with SaCas9 and GFP. Tissues were harvested at 1, 2, 4, 6, and 12 weeks post-injection. (B) CD8+ T cells as a percentage of total lymphocytes in the liver were measured by flow cytometry. (C) CD8 mRNA expression between mice immunized against ovalbumin and SaCas9 measured by qPCR. (D) Representative TUNEL staining in mouse liver sections. (E) Serum alanine transaminase (ALT) activity levels indicating liver damage. (F) Representative Ki-67 staining of mouse liver sections. (G) Ki-67 mRNA expression in mouse liver. Group sizes in (C), (E), and (G) are n = 7, except for ovalbumin 6 weeks (n = 6) and SaCas9 1 week (n = 6).CC BY-NC-ND · PubMed Central
Figure 3 Pre-existing Immunity to SaCas9 Results in Elimination of Gene-Edited Hepatocytes following AAV Transduction (A) AAV genome copies of GFP transgene measured via qPCR. (B) Fold change of GFP genome copies relative to ovalbumin-treated mice. (C) AAV genome copies of SaCas9 transgene measured via qPCR. (D) Fold change of SaCas9 genome copies relative to ovalbumin-treated mice. (E) Representative western blots of LDLR, SaCas9 (hemagglutinin, HA-Tag), and GFP in mouse liver. Asterisk indicates non-specific band by the HA-Tag antibody. (F) Ldlr indels in mouse liver samples were measured via deep sequencing.
Figure 3 Pre-existing Immunity to SaCas9 Results in Elimination of Gene-Edited Hepatocytes following AAV Transduction (A) AAV genome copies of GFP transgene measured via qPCR. (B) Fold change of GFP genome copies relative to ovalbumin-treated mice. (C) AAV genome copies of SaCas9 transgene measured via qPCR. (D) Fold change of SaCas9 genome copies relative to ovalbumin-treated mice. (E) Representative western blots of LDLR, SaCas9 (hemagglutinin, HA-Tag), and GFP in mouse liver. Asterisk indicates non-specific band by the HA-Tag antibody. (F) Ldlr indels in mouse liver samples were measured via deep sequencing.CC BY-NC-ND · PubMed Central
Figure 4 CD8+ T Cell and Treg Responses in SaCas9-Immunized Mice after Treatment with Either AAV-GFP or AAV-CRISPR (A) Mice were immunized with SaCas9 protein 1 week prior to delivery of an AAV vector encoding a gRNA targeting Ldlr with SaCas9 or GFP. Tissues were harvested at 2 weeks post-injection. (B) T cells as a percentage of total lymphocytes in the liver were measured via flow cytometry. (C) CD8+ T cells as a percentage of T cells. (D) Percentage of memory CD8+ to total CD8+ T cells. (E) Percentage of activated CD8+ T cells to total CD8+ T cell population. (F) Percentage of activated memory CD8+ T cells to total CD8+ T cell population. (G) Tregs as a percentage of total lymphocytes in the liver measured via flow cytometry. n = 6 per condition.
Figure 4 CD8+ T Cell and Treg Responses in SaCas9-Immunized Mice after Treatment with Either AAV-GFP or AAV-CRISPR (A) Mice were immunized with SaCas9 protein 1 week prior to delivery of an AAV vector encoding a gRNA targeting Ldlr with SaCas9 or GFP. Tissues were harvested at 2 weeks post-injection. (B) T cells as a percentage of total lymphocytes in the liver were measured via flow cytometry. (C) CD8+ T cells as a percentage of T cells. (D) Percentage of memory CD8+ to total CD8+ T cells. (E) Percentage of activated CD8+ T cells to total CD8+ T cell population. (F) Percentage of activated memory CD8+ T cells to total CD8+ T cell population. (G) Tregs as a percentage of total lymphocytes in the liver measured via flow cytometry. n = 6 per condition.CC BY-NC-ND · PubMed Central

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Authors
Li, A., Tanner, M. R., Lee, C. M., Hurley, A. E., De Giorgi, M., Jarrett, K. E., Davis, T. H., Doerfler, A. M., Bao, G., Beeton, C., & Lagor, W. R.
Affiliations
  • Rice University (US)
  • Baylor College of Medicine (US)
Corresponding author
  • Bao, Gang
  • Beeton, Christine
  • Lagor, William R.
ORCID
Venue
Molecular Therapy
Publisher
Elsevier BV
Published
2020-06
Volume
28
Issue
6
Pages
1432-1441
ISSN
1525-0016 (ISSN-L)
DOI
10.1016/j.ymthe.2020.04.017
PMC
PMC7264438
OpenAlex
W3016959969
Cited by
230
References
40
Citations by year
2026: 26 · 2025: 33 · 2024: 33 · 2023: 36 · 2022: 38 · 2021: 43 · 2020: 19
FWCI
11.02
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Therapy (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Digital Commons-TMC (Texas Medical Center) (submittedVersion)
  • Figshare (submittedVersion, cc-by)
  • Health Research Alliance (cc-by)
Funders
  • American Heart Association (19POST34430092, 19PRE34380467, GM08231, HL007676, RR024574)
  • Cancer Prevention and Research Institute of Texas (RR140081, RP180672)
  • National Institutes of Health (RR024574, HL007676, GM08231, CA125123, UG3HL151545, HL132840, U42OD026645)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
Keywords
adeno-associated virus vectors, CRISPR/Cas9, SaCas9, pre-existing immunity, neutralizing antibodies, T cell response, hepatocyte apoptosis, compensatory liver regeneration, SpCas9
MeSH terms
Gene Editing, CRISPR-Associated Protein 9, RNA, Guide, CRISPR-Cas Systems, Dependovirus, Animals, Genetic Vectors, Humans, Immunization, Immunologic Memory, Biomarkers, Gene Expression, Immunophenotyping, T-Lymphocyte Subsets, Transgenes, Hepatocytes, Gene Order, Mice, Clustered Regularly Interspaced Short Palindromic Repeats

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