Gene Therapy and Genome Editing

Zewail City of Science and Technology

  • Establishment and Inauguration:

    • Established in 2000.

    • Inaugurated in 2011.

  • Tagline: "مصر تستطيع" (Egypt Can).

The Human Genome and Disease

  • Focus: Different Approaches for Treatment of Genetic Diseases.

  • Speaker: Dr. Radwa Ayman Salah.

Gene Therapy and Genome Editing Strategies

  • CRISPR/Cas9 System:

    • A prominent gene-editing technology allowing precise DNA modifications in organisms.

    • Originates from bacteria's natural defense mechanisms against viruses.

    • Popularity in Labs:

    • Noted for its simplicity, efficiency, and versatility.

    • Mechanism Details:

    • Composed of two main components:

      • Cas Protein:

      • An endonuclease enzyme responsible for cutting DNA.

      • Activated upon gRNA binding to target DNA.

      • Guide RNA (gRNA):

      • A short RNA sequence tailored to pair with specific target DNA sequences.

      • Contains two sequences:

        • Complementary Sequence: Targets the DNA.

        • Spacer Sequence: Guides the Cas protein to the target location.

    • Process of Editing:

    • gRNA attaches to complementary DNA sequence.

    • Cas protein is recruited to cut DNA, followed by cellular repair mechanisms.

Historical Highlights in Gene Editing

  • Nobel Prize in 2020:

    • Awarded to Emmanuelle Charpentier and Jennifer Doudna for their discoveries in DNA manipulation via the CRISPR-Cas9 system, referred to as "genetic scissors."

New Gene Editing Tools

  • Fanzor:

    • An eukaryotic programmable RNA-guided endonuclease.

    • Operates by cleaving DNA at specific sites guided by RNA molecules.

    • Induces double-strand breaks, activating cellular repair mechanisms useful in genome editing features.

  • NICER Tool (Nicking Induced by CRISPR/Cas9 for Homologous Recombination):

    • Employs multiple nicks created by Cas9 nickase to enhance homologous recombination for correcting heterozygous mutations in somatic cells.

    • Mechanism:

    • Uses homologous chromosomes as repair templates.

    • Success reliant on multiple nicks which increase gene correction efficacy.

Delivery Systems in Gene Editing

  • Goal:

    • Achieve maximum efficiency for therapeutic gene delivery through plasma membrane, cytoplasmic trafficking, and nuclear entry.

  • Vectors for Gene Editing:

    • Essential transportation vehicles for therapeutic materials into cells, divided into viral and non-viral types.

Characteristics of Ideal Vectors

  • Attributes:

    • Non-toxic.

    • Non-immunogenic.

    • High delivery efficiency.

    • Adequate nucleic acid loading capacity.

    • Suitable tissue tropism to avoid excessive spread.

Non-Viral Vectors

  • Types:

    • Bacterial Plasmid DNA (pDNA):

    • Circular DNA vectors providing essential traits to host cells such as drug resistance.

    • Advantages:

      • Easy and inexpensive to produce.

    • Limitations:

      • Immune response triggered due to bacterial sequences.

    • Minicircle DNA:

    • Streamlined plasmids lacking immunogenic bacterial sequences.

    • Features:

      • Higher expression of transgenes and lower toxicity.

    • Mini-Intronic Plasmid DNA (MIP):

    • Newer technology that aims to mitigate traditional plasmid limits.

    • Essential bacterial elements are placed in an engineered intron to enhance gene expression.

Viral Vectors

  • Characteristics:

    • Most effective for specific cell modification.

    • Protect therapeutic genes within their viral casing for effective delivery.

  • Types:

    • Integrating Vectors:

      • Merge genetic material into host genome, preferred for dividing cells.

      • Risk of safety issues as expression can be silenced over time.

    • Non-Integrating Vectors:

      • Remain episomal, low risk of genotoxicity, provide stable expression in non-dividing cells.

Case Studies in Gene Therapy

  • ADA-SCID (Bubble Boy Syndrome):

    • A rare genetic condition where immune system has no B and T lymphocytes due to a deficiency of Adenosine Deaminase (ADA).

    • 1990 marked the first clinical trial using retroviral delivery to treat patients.

    • Long-term results showed varied healing in treated patients after cell infusion.

  • HIV Treatments:

    • 2014: First gene-editing during HIV treatment using ZFN.

    • 2022: CRISPR/Cas9 EBT-101 therapy shows promise with no serious side effects.

  • Leber Congenital Amaurosis (LCA10):

    • 2019: First in-vivo CRISPR-Cas9 trial used to treat retinal disease caused by CEP290 mutations. Results monitored over time for therapeutic impact.

  • Hemophilia B:

    • HEMGENIX® approved in 2022 as a one-time AAV viral vector gene therapy to enable functional Factor IX production.

CAR T-Cell Immunotherapy

  • Process Overview:

    1. T cells collected from patient.

    2. Genetically engineered to target cancer cells.

    3. The modified cells multiplied and re-infused into the patient.

  • Significance:

    • Represents a revolutionary approach in cancer treatment.

Limitations and Challenges of Gene Therapy

  • Issues:

    • Off-target effects, deaths during trials, host immune responses, unpredictable gene expression outcomes.

    • Large therapeutic genes pose transfer difficulties.

  • Targeted Insertion:

    • Use of "safe harbor" loci for higher expression levels.

Conclusion

  • Future Outlook:

  • Personalized gene editing therapies are anticipated to become standard treatments for numerous diseases as advancements continue.

Questions and Further Resources

  • Numerous publications and clinical studies provide additional insights into gene therapy developments. Interested readers can explore provided references and links for more context.