week 9 and week 10a: gene therapy

describe the different levels of gene therapy

what is gene therapy

manipulation of genes to cure a disease; introduction of therapeutic gene into
patient’s cells
● adding DNA to the genome
● altering the DNA of a gene

therapeutic gene/DNA

a gene/DNA whose expression will fight disease

biologics

Gene therapy products are a type of biologics (any pharmaceutical drug product manufactured in, extracted from, or semi-synthesized from biological sources; e.g. from blood, proteins, viruses, living organisms)

• e.g. insulin

somatic cell gene therapy

DNA is transferred/edited in body tissues. Does not prevent the disease
from occurring in the next generation because it does not affect the germline. Many clinical trials and approved somatic cell gene therapy products

• just fixes pts DNA, DNA is not passed down to next gen

germline gene therapy

genetic modification of germ cells. No country permits heritable human
genome editing in clinical practice; some countries allow use of genetically modified in vitro embryos in laboratory research. Criminal offence in Canada

• gets past down to next gen

• can eradicate genetic disease from population

• unethical because can make designer humans

what does gene therapy require?

knowledge of target gene and cell/tissue, knowledge of gene function and disease
mechanism

• need to know the type of mutation, what it is, where it is and what caused it

list and describe the two approaches of gene therapy

• in vivo (inside the body): direct delivery into target organ (tissue easily accessible, cells hard to remove)

• ex vivo (outside the body; cell therapy): removing cells from a patient, modifying them in a culture, reintroducing cells into the patient

what are the basic steps of gene therapy

• accurate diagnosis of the genetic defect/identification of DNA sequence

• therapeutic DNA obtained using PCR, recombinant DNA tech, synthesis.

• DNA is packaged into vector (gene delivery system)

• vector is directly injected in target tissue or added to patient’s cells in culture

• if ex vivo approach, cells are transplanted into patient

• host’s machinery used to express new gene

what are the effects of gene therapy in terms of symptoms

• May provide a longer-lasting effect than treating symptoms or supplying a protein/molecule.

• Unclear if it provides a long-term cure

compare genome editing vs silencing

describe CRIPSR

what is the major difference between ZFN/TALEN and CRISPR/Cas9?

ZFN and TALEN: protein-DNA system whereas CRISPR/Cas: RNA-DNA system

• ZFN/TALEN: protein is specifically designed to recognize the target DNA and bring the nuclease to it

• CRISPR/Cas9: RNA guide recognizes the target DNA and brings the nuclease

name the genome editing nucleases

• what do they do?

Genome editing nucleases (ZFNs, TALENs and CRISPR/Cas9) induce DSBs at targeted sites. DSBs can be repaired by NHEJ or, in the presence of donor template, by HDR

compare the gene editing methods for

• size

• nucleases

• target sequence

• specificity

• limitations

• difficulties engineering

• difficulties delivering

describe gene silencing by RNA interference

explain ribozymes’ gene silencing

• catalytic RNA molecules that recognize their target RNA in a sequence specific manner

explain antisense oligonucleotides’ gene silencing

name and describe the types of nonviral physical methods of DNA transfer

• how each works

describe lipid nanoparticles (LPN) and how they are used to transfer DNA into cells

name and describe the types of viral methods/vectors of DNA transfer

• how they work

• limitations

compare the 4 viral vectors for

• size

• genome

• packaging capacity

• transduction

• integration

• expression

• immunogenicity

• gene therapy strategy

compare allogenic and autologous gene therapy

• allogenic: cells for gene therapy are from a matched donor then injected into pt

• autologous: pts own cells are used for gene therapy

  • cells undergo gene therapy then are reintroduced to pt

  • no chance of the body rejecting the cells

what is Beta-thalassemia?

Autosomal recessive disorder; decreased production of hemoglobin

• due to mutation in Beta hb chain

• causes excess of alpha chains that aggregate and cause damage/anemia

describe the gene therapy for Beta-thalassemia

what is Casgevy

• Casgevy (Vertex Pharmaceuticals Incorporated (Nasdaq: VRTX) and CRISPR Therapeutics)

• For treatment of sickle cell disease and transfusion-dependent beta-thalassemia

  • first approved CRISPR gene editing treatment

  • can use the same treatment for sickle cell disease and transfusion-dependent beta-thalassemia because they have the same gene mutated

• ex vivo CRISPR/Cas9 gene-edited therapy, in which a patient’s own hematopoietic stem cells are edited to produce high levels of fetal hemoglobin (HbF; hemoglobin F) in red blood cells.

• Disrupts the BCL11A gene in red blood cell precursors in the bone marrow. Disruption of BCL11A reactivates the production of HbF (mimicing the HPFH phenotype).

  • get fetal Hb

what is Lyfgenia by BluebirdBio

• At the same time also approval of gene therapy by BluebirdBio; Lyfgenia; $3.1 million; MOA: addition of Hb gene.

• By September 2024: ~10 patients on Casgevy therapy, ~20 on Lyfgenia

• More than 80% of U.S. patients are not suitable for the therapies, which are approved only for those over 12 with a history of severe pain crises. ("You have to be sick, but not too sick," Dr. Andrew Campbell, director of the Children's
  National Comprehensive Sickle Cell Disease Program, Washington D.C)

  • need chemo first, then transplant

  • cost is high

how many approved gene therapies are there globally? RNA therapies?

30 each

describe the current situation of gene therapy clinical trials

• what stages are they in for the most part?

• in vivo or ex vivo

• most are ex vivo

• most gene therapies are currently in preclinical phase (animal models)

• very few get to pre-registration (submitting to FDA)

what are the majority of current gene therapy clinical trials and approved therapies trying to find a cure for?

cancer

describe CAR T-cell therapy