L18 - Gene Therapy II

Biotech Exam II

What are liposomes?

• lipid bilayer formed in a sphere

• cationic, used to transfer negatively charged DNA into cells

Disadvantages of lipisomes

• low efficiency of transfer

• foreign gene does not integrate into host genome

• high liposome and DNA concentrations required

What are PEGylated liposomes?

liposomes with improved half-life

What are transferrin-conjugated liposomes?

contains transferrin that can bind to transferrin receptors found on human cells

What are antibody-modified liposomes?

liposome with antibody coating for specificity

What are sendai (HVJ) viral proteins?

can be added to liposome to allow DNA to escape the endosome into the cytosol intact by disrupting endosomal membrane

What are nanoparticles?

• polymer (PGA or PLGA) + DNA

• protects the DNA from degradation

What percentage of injected naked DNA is degraded by extracellular nucleases?

99%, need large amounts of DNA

• low gene transfer efficiency and protein expression

How can transduction efficiency of naked DNA be increased?

by electric impulse or gene gun

What is a biolistic gene gun

• uses kinetic energy to deliver nucleic acids inside cells

• 5-10% transduction efficiency

• only used for exposed targets, not for deep tissues

What are advantages of non viral vectors?

• low immunogenicity

• can be made to be non toxic

• easy to synthesize

• potentially targetable

• no limit on plasmid size

• no integration, can be administered as drugs

Disadvantages of non viral vectors

• low transfection efficacy

• no natural tropism, endosomal escape, or nuclear transport mechanisms

What are some clinical applications of non viral vectors?

• cancer

• Parkinson’s disease

• restenosis

• retinal disorder

• cystic fibrosis

• hemophilia genetic defects

• severe combine immune deficiency (SCID)

What is adenosine deaminase deficiency?

• autosomal recessive condition

• selective toxicity to WBCs from accumulation of metabolites of deoxyadenosine

What is X-linked SCID?

• absence of functional T cell and NK cells caused by deficiency in IL2RG gene ( y subunit of cytokine IL2 receptor)

What is cystic fibrosis?

• mutation in CFTR gene that normally codes for channel protein found epithelial cell membranes

• symptoms: impaired lung function, chronic resp. infection, airway inflammation

What disease characteristics are ideal for gene therapy?

• single gene defect

• recessive condition

• pathology location is accessible

• progressive disease that provides a therapeutic window from symptom to treatment

What is hemophilia B?

• x-linked genetic disorder

• abnormal bleeding due to defective coagulation factor IX

• current treatment - factor IX infusion (expensive, needs to be given continuously)

• new treatment - using sc-AAV

What is hemophilia A?

• x-linked disorder, 5x more common than hemophilia b

• caused by mutation on factor VIII gene

• good target for gene therapy as even a low level of secreted protein expression can prevent symptoms

What are other applications of gene therapy?

1. leber’s congenital amaurosis (LCA) - caused by mutation in RPE65 gene that affects the retina

2. parkinson’s disease - cell death in substantia negra region of brain, resulting in lack on dopamine

3. lysosomal storage diseases

What are the three broad approaches to selection of a therapeutic agent in Parkinson’s disease?

1. restoration of dopamine synthesis in the dorsal striatum

2. modulation of activity in the basal ganglia downstream of the striatum

3. modification of disease progression by neuroprotection

Vectors associated with gene therapy for Parkinson’s

• GDNF - nigrostriatal dopamine terminal protection

• Ad-GDNF - protect dopaminergic neurons and imrpove dopamine-dependent behavior

• AAV-2 - transduces neurons within the CNS

What is restenosis?

• re narrowing of a coronary artery after treatment with stenting

• can be prevented by coating stent with PLGA nanoparticles containing VEGF gene

What is CAR-T cell therapy?

• t cells engineered to recognize tumor antigens

Suicide gene therapy

• introduction of viral or bacterial genes into tumor cells, which convert a non toxic prodrug into a toxic one

• non toxic prodrug is administered systemically

advantages of suicide gene therapy

• requires short term gene expression

• safety in clinical trials

• bystander tumor killing via gap-junction delivery of drug

• enhances sensitivity to conventional therapy

Limitation to suicide gene therapy

• limited spatial distribution of gene transfer vectors

• poor gene-transfer efficiency into tumor cells in vivo

• inability to target dispersed tumor cells

What is in vivo CRISPR editing?

the editing of genes directly inside the human body by using lipid nanoparticles to deliver CRISPR components to the diseased area

What is base and prime editing?

allow for precise correction of single-letter genetic mutations without cutting the DNA

What are the two components of CRISPR-Cas9?

1. RNA guided DNA endonuclease Cas9

2. chimeric single guid RNA - derived from crRNA and tracrRNA

CRISPR-cas9 mechanism

1. sgRNA binds to Cas9 and directs it to locus of interest via base pairing to the genomic target

2. Cas9 binds to PAM sequence on DNA

3. Two nuclease domain of Cas9 → leads to double stranded break

4. homology directed repair or nonhomologous end joining

What are the two nuclease domains of Cas9?

• HNH domain - cuts target strand

• RuVC domain - cuts non-target strand

What is nonhomologous end joining (NHEJ)?

• error prone

• causes insertions or deletions

What is homology directed repair (HDR)?

• requires donor DNA template

• gene insertion, precise mutations

Disadvatages of CRISPR technology

• ethical concerns about germline gene editing

• lack of specificity in targeting and incomplete targeting

• modified organisms could endanger biodiversity

What are potential complications of gene therapy?

• gene silencing - repression of promoter

• genotoxicity

• phenotoxicity - complications from overexpression of gene

• immunotoxicity

• risk of horizontal transmission - shedding of infectious vector into environment

• risk of vertical (germline) transmission