Gene Therapy

What are the steps of gene replacement therapy?

1. Isolate the gene 2. Pack functional gene into viral vector 3. Use the in vivo approach: insert the packaged gene into the patient OR Use the ex vivo approach (most common): put packaged gene into a culture of the patient's cells and then put into patient.

siRNA-Based Drugs • _____ #siRNA drugs approved by FDA since 2018 §treat rare monogenic diseases? transthyretin amyloidosis, acute hepatic porphyria, primary hyperoxaluria type 1 • _____ # siRNA drugs in late stages of phase 3 trials §____________ - for hypercholesterolemia •RNA is modified to make it more stable •two major delivery mechanisms §nanoparticles that pass through membranes §conjugated ligand allowing uptake by receptor mediated endocytosis •annual revenues are expected to rise over the next few years §inclisiran sales expected to reach $1.2 billion by 2024

3, 7, inclisiran

•One dose reduced LDL cholesterol by __________ §effect maintained at least 6 months •Will require only _____ injections/year §better than daily statin dosing §better than anti-PSCK Abs required every 2-4 wks

30-50%, 2-3

•May require expression of only __________ of normal levels for significant health benefit

5-20%

Future human gene therapy is likely to involve gene editing to correct the defective gene within the genome using the CRISPR/Cas9 system. What is the role of Cas9 in this system? A. It generates a double stranded cut in the target DNA B. It cleaves individual guide RNAs from RNA transcripts C. It ligates the ends of the target DNA after it has been cut D. It guides the nuclease to the specific target site in the DNA E. It cuts one strand of the target DNA to allow homologous recombination to occur

A. It generates a double stranded cut in the target DNA

ALD is caused by mutations in ____________ that encodes peroxisomal transporter for VLCFAs •Beta oxidation of VLCFAs only occurs in peroxisomes

ABCD1 gene

_______________________________ Does not integrate into the genome--> episome Confers long term gene expression infect dividing and non dividing cells Package 5 kb DNA

Adeno-associated viral vector (DNA)

Severe Combined Immunodeficiency (SCID) is an autosomal recessive disorder caused by a deficiency in _________________ and is characterized by a lack of T-cells, B- cells and NK-cells (severe lymphopenia) hence no cellular or humoral immunity in these patients which makes them vulnerable to recurrent opportunistic infecctions --> hepatosplenomegaly. Without treatment --> death before age 2. 1-5/million live births

Adenosine Deaminase

•Rare X-linked disorder, 1 in 20,000-50,000 boys •Childhood cerebral form (~35%) - impaired adrenocortical function - progressive neurological deterioration due to demyelination •Symptoms begin at 3-10 years of age (peak at 7) •Total disability within 6-24 months followed by death

Adrenoleukodystrophy (ALD)

Why are ALD and ADA-SCID good candidates for gene replacement therapy? A. Disease severity B. Poor treatment options with exception of HSC transplant (limited availability) C. Stem cells easily manipulated ex vivo and re-infused for systemic delivery D. All of the above E. None of the above

All of the above

A 4-month-old female presents to her pediatrician with fever and cough for a few days, diarrhea for 3 weeks and oral thrush for 3 months. Abnormal physical findings include temperature of 38.3°C, weight <3rd percentile, moderate dehydration, severe oral thrush, and a general ill appearance. No palpable lymph nodes or organomegaly are detected. Chest auscultation shows clear lungs and normal heart sounds. Laboratory findings reveal severe lymphopenia (low B- and T-cell counts) and barely detectable adenosine deaminase (ADA) activity in her red blood cells. What will be the first treatment provided to this patient? A. Treatment with cytokines B. Gene therapy with a replacement ADA gene C. Enzyme replacement therapy with PEG-ADA D. Stem cell transplant using cells from an HLA-matched sibling donor E. Treatment with a bone marrow stimulating drug to boost immune system development

C. Enzyme replacement therapy with PEG-ADA

CRISPR/Cas9 System is composed of: - _______________________ creates double-stranded DNA break - _______________________ directs nuclease to appropriate target DNA

Cas9 Endonuclease, Guide RNA

Treatment for ALD •________________ replacement for adrenal insufficiency •_______________________________________________ - only effective in early stages of disease - donor derived myelo-monocytic cells migrate to CNS and replace diseased microglia - arrests cerebral demyelination -suitable donor available for <25% of patients

Corticosteroid, Allogeneic hematopoietic stem cell transplant

All of these are challenges to gene therapy except: A. Transient or low level expression B. Inefficient delivery to appropriate tissue C. Insertional mutagenesis D. Immune response to vector or gene product E. All of the above F. None of the above

E. All of the above

Which is the following are ways to deliver wild-type gene? A. viral vectors B. plasmids C. naked DNA D. liposomes E. all of the above

E. All of the above

A 7-year-old boy and his parents are seeing his pediatric neurologist for a follow-up appointment. The boy's newborn screening results and follow-up confirmatory genetic testing led to a diagnosis of adrenoleukodystrophy (ALD) shortly after birth. His most recent annual MRI screening has revealed, for the first time, areas of demyelination in the cerebral white matter. He has no other signs or symptoms of disease progression. Hematopoietic cell transplantation has been ruled out because no matched related donor could be identified. The neurologist recommends he enroll in a clinical trial of gene therapy for ALD. How is this therapy delivered? A. Intravenous injection of virus carrying the normal ALD gene B. Correction of the mutated ADA gene in vivo via CRISPR/Cas9 C. Intravenous injection of recombinant human ALD protein (ABCD1) D. Injection of virus carrying the normal ALD gene into cerebral spinal fluid E. Infusion of patient-derived stem cells that have been modified to express the ALD gene

E. Infusion of patient-derived stem cells that have been modified to express the ALD gene

A 60-year-old female with hypercholesterolemia has taken a variety of statin medications over the past few years and made sustained changes to her diet; however, her LDL-cholesterol level has remained >200 mg/dl. Her physician recommends she consider enrolling in a clinical trial of the drug inclisiran, a novel oligonucleotide therapy that has been shown to lower cholesterol levels in patients who have not obtained significant cholesterol reductions on statin therapy. How does this oligonucleotide therapy work? A. It induces exon skipping in the LDL receptor gene B. It increases the expression of the LDL receptor gene C. It induces exon skipping in the HMGCoA reductase gene D. It directs the degradation of the mRNA encoding HMGCoA reductase E. It directs the degradation of the mRNA encoding a regulator of LDL receptor turnover

E. It directs the degradation of the mRNA encoding a regulator of LDL receptor turnover

In early clinical trials of gene therapy for X-linked severe combined immunodeficiency (SCID) several patients developed cancer as a direct result of the gene therapy. What was the mechanism for development of the cancer? A. The gene that was delivered was expressed at too high a level B. The vector carrying the gene therapy induced an immune response C. The gene that was delivered knocked out the expression of a tumor suppressor D. The gene that was delivered was expressed in tissues other than the target tissue E. The gene that was delivered randomly integrated into a site that activated a proto-oncogene

E. The gene that was delivered randomly integrated into a site that activated a proto-oncogene

It is defined as the therapeutic delivery of nucleic acid polymers into a patient's cells as a drug to treat disease.

Gene Therapy

Statin inhibit _________________ and increases ______________________ expression on cell surface

HMG CoA reductase, LDL receptor

_____________________________ •34 million Americans at >240 mg/dL total cholesterol (<200 optimal) •Genetic and environmental risk factors •LDL receptor unable to remove LDL cholesterol from blood effectively •Currently treated with _________________ (>32 million Americans) §inhibit HMG CoA reductase §increases LDL receptor expression on cell surface §some patients experience side effects or show lack of efficacy

Hypercholesterolemia, statin

_________________ made by hepatocytes regulates level of LDL receptor on cell surface by stabilizing it in the lysosome for degradation.

PCSK9

Newly ADA-SCID diagnosed patients are given the enzyme replacement therapy known as ____________ which increases the half life of the ADA enzyme from minutes to days increasing its immunogenicity. It's an intramuscular injection 1-2x/week given up to 2 years only because it loses its efficacy over time. This allows time to set up first line treatment options such as Stem Cell Therapy or Gene Replacement Therapy.

PEG-ADA

How does PEG-ADA work?

PEG-ADA remains extracellular in the body and causes a drop in extracellular adenosine and deoxyadenosine. This causes intracellular adenosine and deoxyadenosine to leave the cell reducing its intracellular concentration as well and as a result it reduces the levels of dATP.

____________________________________________ Stably integrate in genomes of dividing and non-dividing cells Safer with respect to integration sites Package ~8 kb DNA The most popular of which is __________________

Retroviral vectors RNA, lentivirus

RNAi for Gene Therapy Challenges:

Stability of RNA Efficient delivery of dsRNA to appropriate cells Transport of dsRNA into cytoplasm of cells

ADA-SCID treatment options:

Stem cell transplant from HLA matched sibling Gene replacement therapy

•Diagnosis of ALD confirmed by increased plasma ___________ levels and identification of a mutation in his ___________ gene.

VLCFA, ABCD1 (ALD)

Gene Editing with CRISPR/Cas9 •Endonuclease creates ______________ break at specific site in genome •Cell repairs it by: - ____________________ insertions/deletions to make knockouts or restore reading frame -_____________________:gene correction using wildtype template. Can correct mutant gene •"Biggest game changer to hit biology since PCR"

double-stranded, non-homologous end-joining, homology directed repair

Lenti-D Therapy - Outcomes Lenti D therapy appears to be safer than stem cell transplant because no _________________________ is required and ____________________________ disease

immunosuppression, graft vs host disease

Lenti-D Therapy for ALD ________________________ injection to stimulate production of stem cells in the blood ______________________ mild chemotherapy agent is administered to patient to kill off some stem cells to make room for the injection of the modified stem cells Read slide

mobilization, myeloablation

Adenosine Deaminase is involved in _____________________ hence it deficiency results in deoxyadenosine and adenosine accumulation which also causes accumulation of _____________ which is toxic inducing apoptosis of lymphocytes.

nucleotide breakdown, dATP

ADA is a key component of the _________________________ pathway

purine salvage

Gene-blocking/silencing therapy is used for: For gain-of-function or dominant negative disorders For disorders in which downregulating expression of a wt gene can improve symptoms Example: sickle cell disease Downregulate expression of ____________________ fetal Hb expression Increased fetal Hb ameliorates disease severity

repressor that turns off

•2018: Clinical trials for_________________________ and _______________ §Using CRISPR/Cas9 to destroy production of _________, a protein that down-regulates fetal hemoglobin production §Autologous CD34+ stem cells treated ex vivo then infused back into patients •2019: Clinical trial for ___________________________________ §Using CRISPR/Cas 9 to destroy mutation (a cryptic exon) in CEP290 gene that prevents protein production §First trial to use CRISPR/Cas9 in vivo - subretinal injection

sickle cell disease, b-thalassemia, BCL11A, Leber's congenital amaurosis type 10

•First gene therapy in 1990 •In 2016, European Commission approved ________________, an ex vivo stem cell gene therapy

strimvelis

The ___________________ component of inclisirin directs liver specific uptake while the _______________ directs RISC to PCSK9

traintennary GalNAc, guide strand