Genetics E2- Gene therapy

What is the therapeutic delivery of nucleic acid polymers into a patient’s cell as a drug to treat disease?

Gene therapy

Which gene therapy strategy exchanges an abnormal gene for a normal, functional copy?

*modified virus vectors

Gene replacement

What is an example of gene replacement?

Zolgensma for SMA - delivers functional SMN1 gene

Which gene therapy strategy corrects a mutation within an abnormal gene to restore its normal function?

Gene repair

Which gene therapy strategy directly alters the endogenous genomic sequence of a cell to fix or modify the patient's existing DNA?

Gene editing

What is an example of gene editing?

CRISPR for sickle cell (edits BCL11A to inc fetal HgB), ZFNs, TALENs, prime editors

Which gene therapy strategy introduces a new gene into cells to provide a new function or therapeutic benefit?

Gene addition

Which gene therapy strategy alters the level of expression of a particular gene, controlling how much protein a gene makes w/o changing the gene itself?

*antisense oligonucleotides

Gene regulation

What is an example of gene regulation?

Antisense oligonucleotides (Spinraza) - modifies splicing to inc protein production in SMA

What is the carrier molecule used as the gene delivery vehicle to deliver the therapeutic gene into the patient’s target cells?

Vector

What is the MC vector?

Viruses that have been genetically altered to carry normal human DNA

How does in vivo gene therapy occur?

Vector is directly injection into patient

How does ex vivo gene therapy occur?

Target cells are removed from the patient followed by a return to the modified autologous cells after gene transfer in the lab

What term refers to the process where foreign DNA is introduced into a cell by a virus or viral vector?

Transduction

How does CAR-T cell therapy work?

Patient’s T-cells are collected & genetically modified outside the body → engineered gene inserted, instructing T-cells to produce a CAR on their surface → enables recognition & killing of cancer cells; “living drug”

*gene addition

What cells do retrovirus infect?

Dividing cells

How do retroviruses work as gene therapy?

Insert DNA copies into packaging cells of the host → produces copies of human genes (modified so can’t replicate itself) → only work in dividing cells

*ex: used to tx brain tumor where nearby brain cells are not dividing

________ are integrated into the host’s genome and can therefore activate proton-oncogene, leading to tumor formation.

Retroviruses

How do adenoviruses work in gene therapy?

Contains double-stranded DNA that can infect dividing & non-dividing cells but are not integrated into host’s genome

What are downsides of adenoviruses used in gene therapy?

Short lifespan so needs re-administration; Only part of genome is removed which can stimulate host’s immune response (worsens with repeated exposure)

How are adenoviruses administered?

IV

What virus used in gene therapy belongs to the parvovirus family but does not cause disease in humans?

AAV

How do adeno-associated viruses (AAV) work in gene therapy?

Can establish long-term gene expression & has minimal pathogenicity → ideal for therapeutic applications

What class of double-stranded DNA viruses infect neurons specifically; and is currently being investigated to insert DNA into inaccessible neurons?

HSV

What virus can enter non dividing cells through pores in the nuclear membrane & stably integrate into the genome?

*ex- HIV

Lentivirus

What are potential pitfalls associated with viral gene therapy?

Transient & low level expression, toxicity, mutagenesis, immune/inflammatory response, viral reactivation, difficulty reaching target, & need for precise regulation

What type of gene therapy is used to treat sickle cell disease?

CRISPR gene editing approach

How is gene therapy used to treat hemophilia A & B?

Factor replacement via gene therapy

How is gene therapy used to treat B-thalassemia?

Restore normal hgb production

What type of gene therapy is used to treat blood cancers?

CAR-T cell therapy

What type of gene therapy is used to treat melanoma?

Oncolytic virus therapy

What type of gene therapy is used to treat multiple myeloma?

Modified immune cell approaches

How is gene therapy used to treat inherited blindness?

Direct gene delivery to the eye (ex: Luxturna in RPE65 deficiency)

What is the gene regulation approach used to treat rare pediatric disease?

Modified gene splicing to inc protein

*ex: Spinraza for SMA

What is zinc finger nuclease (ZFN) used to treat?

HIV: disable CCR5 on T-cells

Metastatic melanoma: edit tumor-infiltrating lymphocytes

What is transcription activator-like effector nuclease (TALEN) used to treat?

Epidermolysis bullosa & used in crops (rice)

What are disadvantages to TALEN?

Larger than ZFNs, restricted to targets w/ 5’ thymine & 3’ adenine

What gene editing therapy used Fokl nuclease to cut DNA?

TALEN

What gene editing therapy uses engineered nuclease (Fokl) fused to zinc finger DNA-binding domains?

ZFN

What gene editing therapy uses a Cas 9 nickase to induce single-stranded breaks in DNA & can generate all possible transition mutations, deletions, and insertions?

Prime editors

Why are prime editors safer?

Doesn’t generate double-stranded breaks

What conditions are prime editors being investigated to treat?

Sickle cell & other blood disorders