L.15 Gene Therapy

What is gene therapy

DNA that get’s introduced to a patient to treat a gneetic disease

What does introducing DNA do in gene therapy?

Contains fencing gene to correct the effects of a disease-causing mutation

Gene therapy uses ____ to do ____.

• Sections of DNA (genes)

• Treat/prevent disease

How do you pick DNA for treatment?

Careful selection to correct effect of a mutated disease-causing gene

Timeline of gene therapy

• 1972 → developed, limited success

• Now → promising treatment, benefits > risks

Examples of diseases treated by gene therapy

• muscular dystrophy

• Cystic fibrosis

Availability/spread of gene therapy

• 400+ active studies

• 12+ drugs on market

Types of gene therapy by cell

• somatic

• Germline

Somatic gene therapy

Transfer of a section of DNA to any cell of the body that doesnt produce sperm or eggs

Do somatic gene therapy effects become hereditary?

No, effects are NOT passed onto patient’s children

Germline gene therapy

Transfer of a section of DNA to cells that produce eggs of sperm

Are the effets of Germline cell therapy hereditary?

Yes, effects will be passed onto to children/ future generations

Gene therapy strategies

• gene augmentation therapy effects

• Gene inhibition therapy

Gene augmentation therapy

• to treat diseases caused by a mutation that stops a gene from producing a fening product (like a protein)

• Makes gene work more

Gene inhibition therapy

• Treat infectious diseases, cancer, and inherited diseases caused by inappropriate (high) gene activity

• Makes gene work less

What happens in gene augmentation therapy?

• add DNA with fenal version of lost gene into cell

• Produce fencing product at sufficient levels to replace protein that was originally missing

Risk of over augmentation of genes

Many disorders don’t require 100% activity to fix → bad outcome

When is gene augmentation therapy successful?

• effects of disease are reversible

OR

• effects of disease have not result in lasting damage

What can gene augmentation therapy treat (diagnosis ex.)

Loss of fen disorders like cystic fibrosis

Many gene therapies have ___ term effects

Short

Bubble boy condition

X-linked form of severe combined immunodeficientcy (X-SCID)

X-SCID is defined by

Mutations in gene of the common gamma chain protein

Effects of X-SCID

Defects in interleukin signalling leads to near absence of T and NK cells, complete lack of B cell function

Less severe SCID

Adenosine delaminate (ADA) deficiency

ADA deficiency can be treated by

Gene therapy

Approach to ADA-SCID therapy?

Retroviral gene therapy

Explain ADA-SCID therapy

• isolate and transducer T cells using a retroviral using a retroviral before that expressed renal ADA

Safety precaution for ADA-SCID treatment

Additional ADA injected weekly → transient success of therapy

Issue with ADA-SCID therapy

• short term effect

• Transferred T cells had no selective advantage, unable to form stable population and died (patient had no side effects)

How is DNA transfer done?

• DNA 1 gene containing instructions fir renal protein packaged in a vector

• Carried into infected cells

• New DNA expressed by cell’s normal machinery

• Produces therapeutic protein/treatment

Example of a vector. Function in therapy

• virus

• Vehicle to carry new DNA into cell

Most DNA transfers are for

Gene replacement

What is an AAV? Is it useful?

• adénome-associated virus

• Currently one of best options for gene therapy, including for CF

Gene causing cystic fibrosis

CFTR

Theoretical advantaged of AAV vector design of CF

• AAV has natural tropism for airway epithial cells (mimics in vivo infection)

• Minimal inflammatory response

• Stable expression

• Does not activate possible oncogenes

• Inserted remains stable and expressed in vivo

Benefit of infection rate of AAV vectors for CF treatment

• infection rate is low

  • Not overloading cells therefore no uncontrolled expression

Why is CF a good candidate for general therapy

• affected gene is known

• Target tissue (lung) is accessible

• Less than 50% gene transfer many confer clinical benefit

Aim of gene inhibition therapy

• introduce a gene whose product

  • Inhibit expression of another gene

OR

• interferes with the activity of the product of another gene

Basis of gene inhibition therapy

Eliminate the activity of a gene that encourages the growth of disease-related cells

Example of gene inhibition therapy

• cancer result of over-activated oncogene (stims cell growth)

• Eliminate oncogene activity to prevent cell growth and stop cancer

How did 2010s improve gene therapy?

• better viral vectors

• Added regualtory elements (promoters/enhancers)

Gene therapy for leukemia

• target SCID-X1

• Modified vectors precisely target expression of genes in specific cell types, don’t go astray, don’t trigger immune system → better success

Different types of vector lifespan

• deliver genes, work short-term, deactivate self

• Deliver genes, work long-term, pass to daughter cells as divide

Popular viruses for gene therapy

• adenoviruses

• Adenovirus-associated viruses

• Lentiviruses

Example of improved vector and use

• lintivarus for sickle cell anemia

• Silencers BCL11A, leads to production of fetal hemoglobin unaffected by sickle cell mutation

Engineering of sickle cell anemia and purpose

• silence gene only in precursors of RBCs

• Treated blood ste cells live LT in bone marrow (reduce, not replace expression)

• Higher specificity optimizes targeting of disease-causing cells/genes

How is precision accomplished in sickle cell anemia treatment

• optimization shRNAs embedded in miRNA (shRNAmir) Architecture

• Achieves ubiquitous (everywhere) Knockdown of BCL11A

Other gene therapies

• RNA interference

• Antisense oligonueleotides (ASOs)

• Messenger RNAs

• Killing specific cells

RNA interference

• old

• Small RNAs to silence targeted gene by neutralizing gene’s mRNA (similar to lentivirus)

Antisense oligonucleotides (ASOs)

• drugs with short, synthetic DNA or RNA

• Target mRNA form faulty gene

• Prevent translation into “bad” protein or trick into making a “good” protein

Messenger RNA treatment

• used for some COV-19 vaccines

• MRNAs introduce genetic code that cells use to make COV-19 spike protein, encouraging development of antibodies

Purpose of therapies that kill cells and how

• suitable for diseases like cancer, that can be treated by destroying certain groups of cells

• Insert DNA that causes cell to die

Ways to insert fatal DNA

1. Inserted DNA contains suicide gene → produces fatal/toxic product (direct)

2. Causes expression of a protein to mark cells so they are attached by body’s natural immune system (indirect)

Important aspect of targeted killing of cells

DNA targeted appropriately to not damage normal cells

Challenges of gene therapy

• delivering to correct place and turning it on

• Avoiding immune response

• Making sure new gene doesn’t disrupt fen of other genes

• Cost

Delivery of gene to right place and activation importance/challenge

• wrong cell = ineffiecent, potential health problems

• Once cell targeted cells can obstruct activation by shutting down genes with unusual activation by shutting down genes w/ unusual activate

avoiding immune response challenge

• new genes can be considered as an “intruder”

• Immune response can be harmful to patient

Not disrupting other genes challenge

• want gene to integrate into genome

• Risk of disruption of healthy cells

Cost challenge

• eligible genetic disorder rare

• Requires individual approach → effective but expensive $$$

Risks of gene therapy

• unwanted immune reaction

• Target wrong cells

• Infection from virus

• Tumor formation

• Off-target effects

Immune reaction risk

Can result in inflammation/organ failure if see introduced vectors as intures

Wrong target risk

• virus can affect multiple cell types → not just mutated

• Can damage healthy cells

  • Cause other disease

Viral infection risk

Virus could recover ability to cause disease

Tumor risk

Wrong spot of insertion, can lead to tumor

Off-target effects risks

• wrong genes/cells targeted

• Result in unanticipated activity

CAR-T Therapy

• chimeric antigen receptor T cells

• Use own immune cells to fight cancer

How does CAR-T work?

• interact T cells

• Engineer/alter it destroy cancer cells

• Infused back into body

CAR-T benefit

Own cells → lower chance of immune response

CAR-t use and success rate and example

• testing in a few lethal cancers as last resort, some success →remission

• Ex-bone marrow cancer

Describe gene editing

• cut and paste changes to genome

• Very specific, precisely target problem genes

• Cut/break in DNA, knock out faulty gene, insert new or both

Best gene editing system

CRISPR/Cas9 therapy

Delivery of CRISPR therapy

• CRISPR/Cas9 (enzyme) or RNP (RNA and protein) complex into delivery vehicles

• Edits ex vivo or in vivo

EX vivo editing

• extract target cells

• Cell culture and expression in vitro

• Delivery of CRISPR for edits selection and expression

• Reintroduce cells to patient

In vivo edits

• via IV infusions

• CRISPR through blood to target OR

• Local with injections to target tissue

What is mitigated with CRISPR

Manny issues/challenges/risks with gene therapy

Why is gene therapy last resort

High risks

Gene therapy next steps

• gene editing

• Universal donor cells

• Personalized therapy

• Common disease