Concise Notes on Gene Therapy

Gene Therapy Overview

  • Definition of Gene Therapy: Introduction of nucleic acids into cells for therapeutic effect, replacing missing or defective genes.
  • Targeting genetic disorders associated with a faulty gene.

Rare Diseases

  • Definition: Affecting fewer than 200,000 people in the US or 1 in 2000 in Europe.
  • Gene therapy is a high prospect due to the genetic etiology and lack of treatments.
  • 80% are caused by faulty genes, often chronic or life-threatening.
  • Many result in premature deaths in infants/children or are fatal in early adulthood.

Therapeutic Strategy

  • Replace faulty gene with corrected version or silence over-expressed gene.
  • Most rare disease genetic disorders are monogenetic.
  • Germline mutations lead to genetic diseases, while somatic mutations lead to cancer.

Delivery Issues

  • Nucleic acids require a vector for cell entry and protection from nucleases.
  • Vectors can be viral or non-viral.

Viruses as Delivery Systems

  • Viruses naturally introduce their genome into host cells.

  • Viral life cycle involves:

    • Introducing viral genome.
    • Expressing viral proteins.
    • Replicating viral genome.
    • Assembling new virions.
    • Leaving host cell.

Viral Taxonomy

  • Viruses classified by replication mechanism, structure, and genome type (RNA or DNA).

Viral Life Cycle

  • Attachment, penetration, un-coating, biosynthesis, assembly, and release.

Viral Structure

  • Composed of:
    • Envelope (optional).
    • Capsid.
    • Viral genome.

Viral Shapes

  • Helical, polyhedral, spherical, or complex.
  • Size limits genome capacity, affecting gene therapy vector development.

Viral Serotypes

  • Variations within a viral species, affecting tissue-specific targeting.

Viruses as Vectors

  • Viral genome replaced with a gene of interest (GOI).

Producing Recombinant Proteins

  • Utilize plasmid DNA to induce recombinant protein production in transfected cells.
  • For viral vectors, recombinant proteins are viral proteins.
  • Host cell is typically of mammalian origin.

Recombinant Virus Production

  • Incorporate viral genome into plasmids.
  • Use separate plasmids to prevent wild-type genome replication.
  • Create a plasmid containing the GOI.
  • Transfect mammalian cells, allow assembly, and harvest GOI-containing viral particles.

Viral Genes

  • Packaging, envelope, and transfer plasmids are co-transfected into mammalian cells.
  • Generational improvements enhance production quality and safety.

Plasmid Design

  • Incorporate signals like Psi (ϕ) for lentivirus and Inverse Tandem Repeats (ITR) for AAV into transfer plasmid.

Viral Vectors: Choices

  • AAV: mild immunogenicity.
  • AV: high transduction efficiency but transient expression.
  • Retro/Lentivirus: stable expression, integrates into the host genome.

Integrating vs Non-Integrating Vectors

  • Retroviral vectors integrate into the host genome for permanent expression.

Retroviral Insertional Mutagenesis

  • Integration can activate or silence genes, posing risks.

SIN Vector Design

  • Self-inactivating (SIN) vectors remove U3 from the 3’ LTR to prevent promoter activity and insertional mutagenesis.

Adenovirus (AV)

  • Non-enveloped, small virus with dsDNA genome.
  • Capsid composed of hexons and pentons.

Adenovirus Life Cycle

  • Phasic replication cycle with early and late phase proteins.

Adenovirus Genome

  • Utilizes both DNA strands, employs alternate splicing.
  • Inverted terminal repeats essential for packaging and transcription.

Adeno-Associated Virus (AAV)

  • Small, non-enveloped virus with ssDNA genome.
  • Helper-dependent virus, requires AV or herpesvirus for replication.

AV & AAV Production

  • Transfer plasmids require ITRs flanking the GOI.

Viral Vector Production Process

  • Cell expansion, transfection, virus production, recovery, purification, and formulation.

Viral Vector Transduction

  • Efficiencies described by transduction units, efficiency, and multiplicity of infection.

Vectors in Clinical Evaluation

  • Adenovirus, retrovirus, plasmid DNA, AAV, and lentivirus are commonly used.

Viral Vectors: Clinical Evaluation

  • AAV gaining traction due to low immunogenicity and good serotype-specific tropisms.

Luxturna

  • First gene therapy approved by FDA (2017).
  • Targets Leber Congenital Amaurosis (LCA) by using AAV to deliver RPE65 gene.

Development of Glybera

  • First human gene therapy product for lipoprotein lipase deficiency (LPLD).
  • Illustrates challenges in translational research and business planning.

Summary

  • Gene therapy development requires multiple levels of expertise.
  • Consultation and collaboration are vital for success.