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

Literature Information ​

DOI10.1016/j.ymthe.2020.04.017
PMID32348718
JournalMolecular therapy : the journal of the American Society of Gene Therapy
Impact Factor12.0
JCR QuartileQ1
Publication Year2020
Times Cited117
KeywordsAAV-CRISPR, CD8+ T cell, SaCas9, adeno-associated virus, gene therapy
Literature TypeJournal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.
ISSN1525-0016
Pages1432-1441
Issue28(6)
AuthorsAng Li, Mark R Tanner, Ciaran M Lee, Ayrea E Hurley, Marco De Giorgi, Kelsey E Jarrett, Timothy H Davis, Alexandria M Doerfler, Gang Bao, Christine Beeton, William R Lagor

TL;DR ​

This study investigates the impact of pre-existing immunity to the SaCas9 protein on the efficacy of AAV-mediated CRISPR-Cas9 genome editing in the liver of mice. While efficient genome editing was achieved despite the immunity, it triggered a significant CD8+ T cell-mediated cytotoxic response leading to hepatocyte apoptosis and loss of edited cells, highlighting critical safety concerns for in vivo genome editing applications.

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AAV-CRISPR · CD8+ T cell · SaCas9 · adeno-associated virus · gene therapy

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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Primary Questions Addressed ​

  1. What alternative strategies could be employed to overcome pre-existing immunity to Cas9 in gene editing applications?
  2. How do different AAV serotypes affect the efficiency of CRISPR-Cas9 delivery in the presence of pre-existing immunity?
  3. What are the implications of CD8+ T cell responses for the long-term safety of CRISPR-Cas9 therapies in humans?
  4. Could other CRISPR orthologs or engineered variants of Cas9 be more effective in individuals with pre-existing immunity?
  5. How does the immune response to Cas9 influence the overall therapeutic outcomes in clinical settings for gene editing?

Key Findings ​

Research Background and Objective ​

Adeno-associated viral (AAV) vectors are widely used for delivering CRISPR-Cas9 for therapeutic genome editing. However, the persistent expression of Cas9, a bacterial protein, raises concerns about immune responses in humans. This study investigates the impact of pre-existing immunity to Staphylococcus aureus Cas9 (SaCas9) on AAV-CRISPR-mediated genome editing in the liver, assessing both efficacy and safety.

Main Methods/Materials/Experimental Design ​

The study utilized a mouse model to examine the effects of pre-existing immunity on AAV-CRISPR delivery. Key components of the experimental design included:

  1. Immunization: Mice were immunized with SaCas9 protein or ovalbumin as a control.
  2. AAV Vector Administration: One week post-immunization, mice received AAV vectors carrying a gRNA targeting the low-density lipoprotein receptor (Ldlr) gene and a GFP marker.
  3. T Cell Analysis: Flow cytometry was employed to assess T cell populations in the liver, focusing on CD8+ cytotoxic T cells.
  4. Liver Function Assessment: Liver damage was evaluated through alanine transaminase (ALT) activity and histological analysis.
  5. Genome Editing Evaluation: The efficiency of genome editing was measured by quantifying indel mutations in the Ldlr gene via deep sequencing.

The experimental workflow can be visualized as follows:

Mermaid diagram

Key Results and Findings ​

  • Immune Memory Induction: Mice immunized with SaCas9 developed a robust CD8+ T cell response upon subsequent AAV-CRISPR administration, indicating strong immune memory.
  • Liver Damage: The presence of CD8+ T cells correlated with hepatocyte apoptosis and elevated ALT levels, indicating liver damage.
  • Genome Editing Efficiency: Although AAV-CRISPR transduction occurred, the editing efficiency significantly decreased in pre-immunized mice, with only 3.4% detectable indels compared to 20.5% in controls.
  • Liver Regeneration: Despite the elimination of gene-edited hepatocytes, the liver showed compensatory regeneration.

Main Conclusions/Significance/Innovation ​

The study demonstrates that pre-existing immunity to SaCas9 significantly hampers the efficacy of AAV-CRISPR-mediated genome editing in the liver by eliciting a strong cytotoxic T cell response that eliminates edited cells. This finding underscores the need to consider anti-Cas9 immunity in the design of clinical trials and highlights the potential risks associated with CRISPR-based therapies in individuals with prior exposure to Cas9 proteins.

Research Limitations and Future Directions ​

  • Limitations: The study primarily focused on the liver and may not fully represent immune responses in other tissues. The results were derived from a mouse model, which may not directly translate to human physiology.
  • Future Directions: Further research is necessary to explore the immune response in different tissues and to evaluate alternative CRISPR systems or strategies to evade immune recognition, such as using less immunogenic Cas9 variants or transient delivery methods.
SectionSummary
Research BackgroundInvestigates the impact of pre-existing immunity to SaCas9 on AAV-CRISPR-mediated editing.
MethodsImmunization, AAV vector delivery, T cell analysis, liver function assessment, genome editing evaluation.
Key FindingsStrong CD8+ T cell response, liver damage, decreased editing efficiency, liver regeneration.
ConclusionsPre-existing immunity poses significant barriers to effective AAV-CRISPR genome editing.
Limitations & Future DirectionsFocused on liver; need for broader tissue studies and alternative CRISPR strategies.

References ​

  1. Nucleic acid cleavage with a hyperthermophilic Cas9 from an uncultured Ignavibacterium. - Stephanie Tzouanas Schmidt;Feiqiao Brian Yu;Paul C Blainey;Andrew P May;Stephen R Quake - Proceedings of the National Academy of Sciences of the United States of America (2019)
  2. Pre-existing anti-adeno-associated virus antibodies as a challenge in AAV gene therapy. - Vedell Louis Jeune;Jakob A Joergensen;Roger J Hajjar;Thomas Weber - Human gene therapy methods (2013)
  3. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. - Chengzu Long;Leonela Amoasii;Alex A Mireault;John R McAnally;Hui Li;Efrain Sanchez-Ortiz;Samadrita Bhattacharyya;John M Shelton;Rhonda Bassel-Duby;Eric N Olson - Science (New York, N.Y.) (2016)
  4. Virus infection, antiviral immunity, and autoimmunity. - Daniel R Getts;Emily M L Chastain;Rachael L Terry;Stephen D Miller - Immunological reviews (2013)
  5. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. - Mohammadsharif Tabebordbar;Kexian Zhu;Jason K W Cheng;Wei Leong Chew;Jeffrey J Widrick;Winston X Yan;Claire Maesner;Elizabeth Y Wu;Ru Xiao;F Ann Ran;Le Cong;Feng Zhang;Luk H Vandenberghe;George M Church;Amy J Wagers - Science (New York, N.Y.) (2016)
  6. Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant. - Jenny McIntosh;Peter J Lenting;Cecilia Rosales;Doyoung Lee;Samira Rabbanian;Deepak Raj;Nishil Patel;Edward G D Tuddenham;Olivier D Christophe;John H McVey;Simon Waddington;Arthur W Nienhuis;John T Gray;Paolo Fagone;Federico Mingozzi;Shang-Zhen Zhou;Katherine A High;Maria Cancio;Catherine Y C Ng;Junfang Zhou;Christopher L Morton;Andrew M Davidoff;Amit C Nathwani - Blood (2013)
  7. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice. - Yang Yang;Lili Wang;Peter Bell;Deirdre McMenamin;Zhenning He;John White;Hongwei Yu;Chenyu Xu;Hiroki Morizono;Kiran Musunuru;Mark L Batshaw;James M Wilson - Nature biotechnology (2016)
  8. CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. - Yanni Lin;Thomas J Cradick;Matthew T Brown;Harshavardhan Deshmukh;Piyush Ranjan;Neha Sarode;Brian M Wile;Paula M Vertino;Frank J Stewart;Gang Bao - Nucleic acids research (2014)
  9. A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing. - Ang Li;Ciaran M Lee;Ayrea E Hurley;Kelsey E Jarrett;Marco De Giorgi;Weiqi Lu;Karol S Balderrama;Alexandria M Doerfler;Harshavardhan Deshmukh;Anirban Ray;Gang Bao;William R Lagor - Molecular therapy. Methods & clinical development (2019)
  10. A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing. - Alireza Edraki;Aamir Mir;Raed Ibraheim;Ildar Gainetdinov;Yeonsoo Yoon;Chun-Qing Song;Yueying Cao;Judith Gallant;Wen Xue;Jaime A Rivera-Pérez;Erik J Sontheimer - Molecular cell (2019)

Literatures Citing This Work ​

  1. Immunity to Cas9 as an Obstacle to Persistent Genome Editing. - Veronica Gough;Charles A Gersbach - Molecular therapy : the journal of the American Society of Gene Therapy (2020)
  2. Ready for Repair? Gene Editing Enters the Clinic for the Treatment of Human Disease. - Martijn P T Ernst;Mike Broeders;Pablo Herrero-Hernandez;Esmee Oussoren;Ans T van der Ploeg;W W M Pim Pijnappel - Molecular therapy. Methods & clinical development (2020)
  3. New Directions in Pulmonary Gene Therapy. - Amber Vu;Paul B McCray - Human gene therapy (2020)
  4. Translating CRISPR-Cas Therapeutics: Approaches and Challenges. - Lavina Sierra Tay;Nathan Palmer;Rebecca Panwala;Wei Leong Chew;Prashant Mali - The CRISPR journal (2020)
  5. Delivery Approaches for Therapeutic Genome Editing and Challenges. - Ilayda Ates;Tanner Rathbone;Callie Stuart;P Hudson Bridges;Renee N Cottle - Genes (2020)
  6. Tools for experimental and computational analyses of off-target editing by programmable nucleases. - X Robert Bao;Yidan Pan;Ciaran M Lee;Timothy H Davis;Gang Bao - Nature protocols (2021)
  7. Design of efficacious somatic cell genome editing strategies for recessive and polygenic diseases. - Jared Carlson-Stevermer;Amritava Das;Amr A Abdeen;David Fiflis;Benjamin I Grindel;Shivani Saxena;Tugce Akcan;Tausif Alam;Heidi Kletzien;Lucille Kohlenberg;Madelyn Goedland;Micah J Dombroe;Krishanu Saha - Nature communications (2020)
  8. CRISPR/Cas9 gene editing for curing sickle cell disease. - So Hyun Park;Gang Bao - Transfusion and apheresis science : official journal of the World Apheresis Association : official journal of the European Society for Haemapheresis (2021)
  9. CRISPR-Cas9 gene editing of hepatitis B virus in chronically infected humanized mice. - Daniel Stone;Kelly R Long;Michelle A Loprieno;Harshana S De Silva Feelixge;Elizabeth J Kenkel;R Matt Liley;Stephen Rapp;Pavitra Roychoudhury;Thuy Nguyen;Laurence Stensland;Rossana Colón-Thillet;Lindsay M Klouser;Nicholas D Weber;Connie Le;Jessica Wagoner;Erin A Goecker;Alvason Zhenhua Li;Karsten Eichholz;Lawrence Corey;D Lorne Tyrrell;Alexander L Greninger;Meei-Li Huang;Stephen J Polyak;Martine Aubert;John E Sagartz;Keith R Jerome - Molecular therapy. Methods & clinical development (2021)
  10. Cas9-directed immune tolerance in humans-a model to evaluate regulatory T cells in gene therapy? - Dimitrios Laurin Wagner;Lena Peter;Michael Schmueck-Henneresse - Gene therapy (2021)

... (107 more literatures)

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