4. To Discuss
• Gene therapy
• History of CRISPR
• Mechanism of CRISPR Cas 9 Technology
• Advantages of CRISPR
• Limitations of CRISPR
• Overcoming the Limitations
• Delivery of CRISPR gene therapy
• Application
• Summary 4
6. Why shift from traditional gene therapy to CRISPR
1. Immunotoxicity
2. Oncogenesis
• Examples :
1. Clinical trial ( in US ) non-mutated OTC ( Ornithine trans Carbamoylase ) gene was
delivered to the liver hepatic artery injection of the recombinant adenoviral vector
housing the therapeutic gene Jessie a 18 yr old with a mild form of OTC deficiency
participated in the trial 4 days after the trial he died ( Immunotoxicity from the
adenovirus vector )
6
7. 2. Gene therapy trial
Use of retroviral vectors
Ex vivo delivery of therapeutic transgenes to autologous s CD34+ hematopoietic stem cells
5 Patients developed therapy related leukemia
( Integration of therapeutic gene into LMO2 protooncogene )
7
16. Off target effects
• Off target effects : Unintended mutations at the site other than target
• Factors causing the off target effects :
1. Excess affinity between Cas 9 and the target DNA
2. Features in the sgRNA :
• Seed sequence : 10-12 bp region proximal to PAM ( Protospacer adjacent motif )
• GC content
3. Repair pathway involved ( NHEJ or HDR )
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17. Overcoming the off target effects
• SpCas9HF-1 variant ( High fidelity )
Introduces mutation to 4 residues involved in direct hydrogen bonding between Cas 9 and the
Phosphate backbone of target DNA
Reduces the excess affinity between Cas 9 and target DNA
• Evo Cas 9 and HiFi Cas 9
Altered amino acid residues in the Rec 3 domain ( Nucleotide recognition )
Increases the specificity to induce double stranded Breaks ( DSB ) 17
18. Overcoming the off target effects cont.…
• Cas9_R63A/Q768A Variant
R 63 A mutation destabilizes the R Loop formation In the presence of Mismatches
Q 768 Mutation increases the sensitivity to PAM Distal mismatches
• Optimize Guide design platforms such as :
1. E-Crisp
2. CRISPR-design
3. CasOFFinder
18
19. Overcoming the DNA damage toxicity
• Crispr induced double stranded breaks :
1. Induce apoptosis rather than the desired gene edit ( p 53 Activation )
2. Large deletions and complex rearrangements
• Method of overcoming the DNA damage toxicity and off target effects is Precise genome
editing
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21. Overcoming Immunotoxicity
1. Using of Non Viral vectors ( Nanoparticles, Exosomes, Liposomes )
2. New Cas 9 Variants with reduced Immunogenic risk like Cj Cas 9 ( Cas 9 from
Campylobacter Jejuni )
21
24. Advantages and Disadvantages of Ex Vivo Delivery
• Advantages :
1. Greater safety ( Patient not exposed to gene altering tool )
2. Technical feasibility
3. Tighter Quality control of the edited cells
• Disadvantages :
1. Survival and retention of in Vivo function of the cells outside the patient
2. Limitation of the method to certain cell type that can survive and be expanded in culture
(hematopoietic stem and progenitor cells (HSPCs) and T cells )
• This therapy provides benefits in Hematological disorder and Cancer immunotherapy24
25. Advantages and Disadvantages of In Vivo delivery
• Advantage :
• Expression of the gene editing toolkit can be controlled to target specific organs
• Disadvantage :
1. Degradation by circulating proteases or nucleases, opsonization by opsonin
2. Cargo must reach the target tissue and bypass the vascular endothelium (preventing
accessibility to larger delivery vehicles (>1 nm diameter).
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27. Disease Gene target CRISPR-Cas 9 mediated
intervention
Metastatic Non small cell lung
cancer
PDCD-1 CRISPR-Cas9 mediated PD-1
knockout-T cells
from autologous origin
Metastatic Renal Cell
Carcinoma
PDCD-1 CRISPR-Cas9 mediated PD-1
knockout-T cells from
autologous origin
Esophageal Cancer PDCD-1 CRISPR-Cas9 mediated PD-1
knockout-T cells from
autologous origin
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28. Covid-19
• PAC-MAN (prophylactic antiviral CRISPR in human cells) is a CRISPR-Cas13-based strategy
as a Therapeutic application against COVID 19
• RNA guided RNA endonuclease activity of Cas13d in human cells to eliminate the SARS-CoV-
2 virus
• CRISPR based detection tools such as SHERLOCK, DETECTR, and FELUDA for COVID
19
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29. Sickle cell anaemia
• Exagamglogene autotemcel ( Exa- cel ) by vertex pharmaceuticals for Sickle cell anaemia (
Stage III clinical trial )
29
30. Summary
• CRISPR is Clustered regular interspaced short palindromic repeats
• It is a bacterial adaptive immune response
• CRISPR locus consist of trRNA ( trans RNA ), Cas ( CRISPR associated endonuclease ), PAM
(Protospacer adjacent Motif ), Spacer ( incorporated viral fragment ) which further transcribe
into crRNA ( CRISPR RNA )
• trRNA and crRNA can be combined to form sgRNA ( single guide RNA ) guides Cas to
cause Cleavage at the desired site ( recognition by PAM )
• It is easy, fast and Highly accurate technology
• Limitations include Off target effects, Immunotoxicity, and DNA damage toxicity
30
31. • Off target effect can be reduced by certain Cas Variants such as SpCas9HF-1 variant, Evo Cas 9 and
HiFi Cas 9, Cas9_R63A/Q768A Variant
• Off target effects and DNA induced toxicity can also be reduced by Precise Gene editing
• Precise gene editing involves three methods : CRISPR/Cas 9 HDR ( Using ssODN ), Base editors (
Adenosine base editors and cytosine base editor ) and Prime editor ( Cas 9n and prime editor guide
RNA ( peg RNA ) )
• Delivery of CRISPR can be by Ex Vivo or In Vivo method
• Delivery agents include Physical method ( Electroporation, Microinjection ), Viral Vectors
(Adenovirus ) and Non viral vectors ( Nanoparticles )
• Applications Include in Cancer, COVID 19 ( Detection ( DETECTR ) and therapeutic ( PAC-MAN ) )
• Exagamglogene autotemcel ( Exa- cel ) for Sickle cell anaemia
31
32. References
• Uddin F, Rudin M, Sen T. CRISPR Gene Therapy: Applications, Limitations, and Implications
for the Future. Front. Oncol.2020;10:1387.
• Zhang XH, Tee LY, Wang XG, Huang QS, Yang SH. Off-target Effects in CRISPR/Cas9-
mediated Genome Engineering. Mol Ther Nucleic Acids. 2015 Nov 17;4(11):e264.
• Zhu Y. Advances in CRISPR/Cas9. Biomed Res Int. 2022;2022:9978571.
• Liu W, Li L, Jiang J, Wu M, Lin P. Applications and challenges of CRISPR-Cas gene-editing to
disease treatment in clinics. Precis Clin Med. 2021;4(3):179-191.
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