THEME 9: GENE THERAPY & VIRAL VECTORS

Definition of gene therapy (EMA)

Medicinal product containing recombinant nucleic acid to regulate, repair, replace, add or delete a genetic sequence

Definition of gene therapy (FDA)

Product mediating effects via transcription or translation of transferred genetic material

In vivo gene therapy

Direct delivery of genetic material into the patient

Ex vivo gene therapy

Cells modified outside the body and then transplanted back

Autologous ex vivo therapy

Patient’s own cells are genetically modified

Allogeneic ex vivo therapy

Cells from another individual of the same species

Xenogeneic ex vivo therapy

Cells from a different species

Main applications of viral vectors

Basic research, gene therapy, vaccines

Use of viral vectors in basic research

Overexpression or knockdown of genes

Diseases targeted by gene therapy

Cancer, monogenic, cardiovascular, infectious and neurodegenerative diseases

Use of viral vectors in vaccines

Immunization by antigen expression

Key safety requirement of viral vectors

Non-replicative or conditionally replicative

Toxicity requirement of viral vectors

Minimal effect on cell physiology

Stability requirement of viral vectors

Long-term expression without genetic instability

Cell specificity requirement of viral vectors

Selective targeting of cell types

Main viral vector families

RNA and DNA viruses

Retrovirus advantage

Genome integration and long-term expression

Retrovirus disadvantage

Insertional mutagenesis and infection of dividing cells only

Lentivirus advantage

Infects dividing and non-dividing cells

Lentivirus disadvantage

Insertional mutagenesis risk

Adenovirus advantage

High titre and wide tissue tropism

Adenovirus disadvantage

Immunogenicity and transient expression

AAV advantage

Non-pathogenic and stable expression

AAV disadvantage

Limited insert size

Vaccinia virus advantage

Large insertion capacity and clinical experience

Vaccinia virus disadvantage

Immunogenicity

Definition of chimeric vectors

Combination of elements from different viruses

Definition of virosomes

Viral genome packaged in a lipid capsid

Tumor tropism of viral vectors

Many target receptors overexpressed in cancer cells

Adenovirus genome type

Double-stranded DNA

Adenovirus early genes

E1A, E1B

Function of E1A

Blocks Rb and forces entry into S phase

Function of E1B-55k

Inhibits p53 and blocks apoptosis

Role of E4-ORF3

Silences p53 target gene promoters

Non-replicative adenovirus

Lacks essential early genes

Adenoviral vector production

Recombinant DNA, packaging cells, viral stock production

Generations of adenoviral vectors

First, second, third generation and gutless

Adenoviral targeting strategies

Capsid modification, genetic and non-genetic targeting

Tumor targeting using promoters

Tumor-specific promoters drive transgene expression

Prodrug activation strategy

Delivery of enzymes converting prodrugs into toxic compounds

Definition of CRADs

Conditionally replicative adenoviruses

CRAD selectivity

Replicate only in tumor cells lacking tumor suppressors

AAV family

Parvoviridae, Dependovirus

Number of AAV serotypes

12

Helper viruses for AAV

Adenovirus, herpesvirus, vaccinia virus

AAV genome components

Rep and Cap genes

Function of Rep proteins

DNA replication, transcription control, integration

Function of Cap proteins

Viral capsid formation

AAV receptor

Heparan sulfate proteoglycans

AAV major advantage

All viral genes removed for safety

AAV major limitation

Small genome limits insert size

Retroviridae genome

ssRNA with reverse transcription

Retrovirus enzymes

Reverse transcriptase, RNase H, integrase

Retroviral structural genes

gag, pol, env

Oncoretrovirus feature

Induces tumors via genome integration

Retrovirus particle size

80–100 nm

Retrovirus genome size

7–12 kb

Lentivirus receptor

CD4 with CCR5 or CXCR4

Lentivirus advantage

Infects non-dividing cells

Lentiviral vector generations

Progressive deletion of viral genes

First approved gene therapy trial

ADA-SCID in 1990

ADA-SCID strategy

Ex vivo modified autologous T lymphocytes

SCID-X1 gene therapy

IL-2 gamma chain in retroviral vector

Major complication of SCID-X1

Insertional mutagenesis causing leukemia

Gelsinger case cause

Immune reaction to adenoviral vector

Outcome of Gelsinger case

Death due to multiorgan failure

Gendicine composition

Adenovirus carrying p53

First cancer approved for gene therapy

Head and neck squamous cell carcinoma

Glybera vector

AAV-LPL

Disease treated by Glybera

Lipoprotein lipase deficiency

HSV-GM-CSF therapy

Oncolytic virus expressing GM-CSF

CAR-T cell therapy principle

Genetically modified T cells targeting tumor antigens

First CAR-T therapy approved

CD19-specific CAR-T

Disease treated by CD19 CAR-T

B-cell acute lymphoblastic leukemia

AAV-RPE65 therapy

Treatment for inherited retinal dystrophy

AAV9-SMN1 therapy

Treatment for spinal muscular atrophy

Most used vectors in clinical trials

Adenovirus, retrovirus, lentivirus, plasmid DNA

Definition of orphan medicine

Treatment for rare life-threatening diseases

Accelerated assessment (EMA)

Faster evaluation of high public health interest drugs

Conditional marketing authorization

Approval with less complete data for unmet needs

Compassionate use

Use of unapproved medicines under strict control