Lecture 7: Genetic Therapies

What are Genomic therapies? what is required for it to be successful?

Nucleic acid base therapies

• These are therapies that allow/promote delivery of genetic material into a patient’s cell

Requires core knowledge of clinical and laboratory principles of medical genetics to be successful

********What are the 3 types of Genomic therapies?

1. Gene therapy: Delivering a DNA sequence into the nucleus, may or may not integrate

2. Genome editing: Deliver a DNA sequence into the nucleus and edit the host’s DNA sequence or integrate the new DNA sequence into a specified location

3. RNA based therapy: Delivering a RNA sequence into the cell. Does not integrate.

*****What are questions to ask when determining Genome therapies?

• 3 categories

*****What is the Basic flow of Gene therapy?

• what is the processes of determining what type of gene therapy?

*****What are the 3 possible Purposes of the chosen therapy?

****What is the purpose of the chosen therapy dictated by?

Genotype + mechanism of disease

*****What are the 2 types of Gene therapy delivery: aka Location that the therapy administered on?

1. TARGET organ/ local = /within a target organ

2. SYSTEMIC = into the blood stream

*******Compare + Contrast TARGETED vs. SYSTEMIC delivery of Therapies

• pros + cons

SYSTEMIC: Achieve broad coverage and expression

• cons: Therapy may NOT get to target organ + HIGHER RISK OF SIDE EFFECTS

TARGETED: Deliver directly into desired organ + decreases risk of unintentional side effects

• Cons: Could be Challenging to ACCESS to DESIRED ORGAN + If the disease has a multisystem, the full disease spectrum will not bey addressed by the new therapy ( aka. if gene is too targeted it won’t be affective enough)

*****What are 3 barriers that systematic delivery of gene therapy may not cross?

1. Blood brain barrier

2. Blood RETINAL barrier

3. BONE + CONNECTIVE TISSUE barrier

******What are 4 modes of Administration of therapies?

1. Intravenous = to the blood (systemic)

2. Intrathecal (brain)

3. Intracerebral (brain)

4. Intra-muscular

*****Gene therapy delivery: what are the 2 types of VECTORS

• packages used to deliver the load

1. VIRUSES: they know hot to get into a human cell, often they are ATTENUATED or modified so they don’t cause viral illnesses

2. NON-VIRAL

******What are the 2 common type of VIRAL VECTORS?

1. AAV (Adeno-associated virus)

2. Lentivirus

********How to Choose a VECTOR = 4 things to keep in mind

1. Package capacity: vectors have limited capacity, some genes = too large; RNA can be much smaller

2. Genome integration + Duration of expression:

3. Target specificity

4. Safety

*****How does INTEGRATION relate to DURATION of gene therapy expression?

Integration = LONGER expression (long life in host)

NON-integration - limited time for expression

*****INTEGRATION carries the RISK of what?

Insertional mutagenesis

depending on where it integrates, not 100% sure where it integrates

********Compare + contrast the 2 types of VIRAL vectors

• AAV + Lentivirus

  • What type of gene transfer

  • Immunogenicity?

  • SIZE OF CASSETTE?

  • Where does gene delivery occur to?

  • INTEGRATION? duration?

AAV

• AAV vectors are an ideal delivery tool for IN VIVO gene transfer.

• The wild-type AAV is a single stranded DNA genome.

• Similar to Wild-type AAV, recombinant AAVs are nonpathogenic and weakly immunogenic.

• AAV vectors can deliver a therapeutic transgene cassette up to 5 kb into the target cell.

• Gene delivery occurs to the target nucleus leading to production of a functional protein.

• The majority of AAV vector genomes do not integrate into the host genome = short expression

LENTIVIRUS

• Most common use has been for EX-VIVO therapy

• Type of retrovirus and thus lentiviral DNA INTEGRATES into the genome = long term

  • Integration is usually in a non-random manner

  • Can transduce postmitotic and quiescent cells (where as some retroviruses require active cell division for successful infection)

• Lentivirus vectors have a larger capacity, ~9 kb

• Immune response is relatively weak (same as AAV)

Both are low immunogenic but does not mean no immune response

*********NON-VIRAL VECTORS

• Similarities + differences with VIRAL vectors

Similarities: also need the same characteristics

• Protecting genetic material

• Transport into cell

• Unpacking of the vector and efficacy

DIFFERENCES:

• These are polymers, lipids, inorganic particles etc.

• LOWER TOXICITY esp. immunogenicity (advantage of circumventing detection by immune system)

*****What is a CON of NON-VIRAL vectors compared to viral?

However, may lack in gene transfer efficiency, gene expression duration and may have their own safety concerns

• Lack millions of years of evolution that selected for the ability to get into host cells

*****What is the most common/popular form of NON-VIRAL vectors?

Lipid nano particles

******Methodology: Compare EX VIVO + IN VIVO

• How do the processes differ?

• PROS + CONS of both

• What type of Viral vector?

• WHICH IS MORE POPULAR AND WHY?

EX VIVO: Retroviruses (or lentiviruses) are used because they allow integration of the target gene into the genome

• Patients' cells are collected -> cultured -> modified with gene therapy (OUTSIDE THE BODY) -> transplanted back to the patient

• PRO: Still needs a vector to get the target gene into the patient’s cells but has much less immune response as patient’s own cells are used

• CON: specific to a patient + Low accessibility. Patient needs to go to a specialized center. Also, its like a modified transplant procedure thus a lot more invasive

IN VIVO: AAV used

• Cells are not extracted and modified “outside of the body”. Target gene is delivered into the target cells (in the body) through local or systemic delivery

• This is a MORE POPULAR method as the medication is not patient specific and medication comes to the patient = MORE ACCESSIBLE

• This also needs a vector

Give an EXAMPLE of a condition where EX-VIVO is used vs. IN VIVO

EX VIVO: Gene therapy for B-thalassemia

IN VIVO: Approved gene therapy for Leber Congenital Amaurosis due to RPE65 leading to vision loss, via an AAV vector

*****GENOME EDITIONG: Possible applications?

1. Removal or correction of a defective gene within the genome

   1. Gene disruption: Silence a pathogenic gene

   2. NHEJ gene correction: Deletion of pathogenic insert

   3. HDR gene correction: Correct a deleterious mutation

2. Insert a corrective gene into a “safe” genomic loci

   1. HDR gene addition: Introduce a therapeutic gene

3. Target a foreign DNA (example: viral genomes)

Are there any APPROVED GENE EDITING THERAPIES?

No approved therapies yet but lots in pipeline

****What is CAR-T

• What is the process (is it done EX or IN vivo?)

• What is the gene used for the therapy?

• How does it work?

  • What is the target of the therapy?

A promising therapy for cancer

• The package (gene) encodes a CAR (chimeric antigen receptor)

• Done EX VIVO and T cells are targeted.

• These transduced T-cells are transferred back in the patient

• These T-cells recognize cancer cells through the cancer specific antigen and would lead to cancer cell death

*****RNA based therapies: Use of RNA molecules offers many different possibilities especially because what?

• It does not integrate into the genome

• Offers a smaller “pay load” (more options of vectors)

• May generate a limited immune response (sort of depends on how it is delivered) - again more vector possibilities

*****What are 2 EXAMPLES of RNA based therapies?

1. ANTISENSE OLIGONUCLEOTIDES (ASO)

2. mRNA

   1. Already have experienced this with covid vacc.

******what are ASO (Antisense Oligonucleotides)

• what do they do?

• What is needed to be understood for ASO to be successful?

Short, synthetic, single (or double) stranded oligonucleotides

Function: Binds a specific RNA sequence of the gene of interest

The mechanism of disease has to be understood to design an ASO approach for a successful therapy

*****What are 2 possible outcomes of ASO after binding gene of interest?

• give an example of each

Modulation of splicing

• Goal could be to restore protein function. In this scenario, the issue would be that low protein expression (or null) leads to symptoms of the disease

  • example: spinal muscular atrophy

RNA knockdown

• Goal could be to limit the expression of a protein. In this scenario, expression of the protein is considered toxic and leads to the disease. Genetic mechanism may be a gain of function (GOF) variant

  • Example: homozygous familial hypercholesterolemia due to PCSK9 variants

*****What are the different types of ASO

• 2 categories

  • 3 total types

Knock down

1. Gapmer ASO

2. siRNA

Splice switching ASO

******GAPMER + siRNA ASO

• what is the outcome/ purpose these ASOs?

• What must we know in relation to the outcome of using these types of ASO? why?

Used to down regulate or knock down a transcript BOTH LEAD TO mRNA DEGREDATION

• Determine if the pathogenic variant leads to a gain of function of a protein

• Must know the downstream effects of downregulation

  • Do low protein levels alter the physiologic state of the cell?

  • Functional studies, examples of biallelic LoF variants in the same gene

  • Example: homozygous familial hypercholesterolemia due to PCSK9 variants

*****Are GAIN OF FUNCTION pathogenic variants typically Auto Dom or Auto recessive disorder?

AUTO DOM

• auto recessive = typically Loss of function

******Splice site ASO

• what are the 2 outcome/ purpose these ASOs?

Create splice site modulation

• Leading to disruption or restoration of READING FRAME

1. Increase protein levels for LoF variants

2. Decrease protein levels for toxic GoF variants

4 Hurdles for gene therapy to become widely used

1. Providing curative/disease modifying treatment for conditions that do not have treatment options

   1. Mostly for monogenic disorders

   2. But some therapies for acquired diseases (cancer) are also a possibility

2. Rare disease populations are small, and accessibility is an issue

   1. Gene therapy in Korea and patient is in Canada

3. Cost

   1. 500K to 2.1 million per treatment

   2. WONT SURVIVE MARKET: Glybera (for lipoprotein lipase deficiency): $1 million. Was withdrawn from the market in 2017 due to high price and low demand

4. Cost and accessibility can significantly reduce the future clinical implementation of these exciting therapies

3 key concerns for the Safety

1. Integration into the genome: With random integration, risks of activating or disrupting nearby genes. This will lead to unintended side effects like cancer, death etc

2. Genome editing could be inaccurate or occur at more than one intended site

3. Immune response: Humans are meant to create an immune response against a virus thus this will either destroy the gene within the virus vector (if patient has been previously exposed to AAV) or will mount an immune response next time, eliminating the possibility of a “second” treatmen

***What are 3 possible SOLUTIONS to random integration

Solutions: combination of techniques

• Site specific integration with genome editing

• Allows insertion of curative gene into a “safe” genomic loci

• RNA based technologies

How is genome therapy EFFECTIVENESS ensured?

Measured via clinical trials

• Phase 1/2/3

4 Medical Genetics contribution in genetic therapies

Medical Geneticists role in Genetic Therapy development

• Preclinical

• Clinical trial

• LTFU