HG exam 3 pgs 31-35

- Availability of tests Right now, so many conditions do not have an approved test

- No current bedside test

Gene therapy

- (def.) introducing cloned genes into living cells to treat disease

- 7000(+) human genetic disorders due to single gene abnormality

- First gene therapy trial in 1989

- 1,700 approved clinical trials since then

- Currently only 2 gene therapies approved by the FDA

- 1) Aug 30, 2017 = Kymriah for acute lymphoblastic leukemia

- 2) Oct 18, 2017 = Yescarta for non-Hodgkin lymphoma

First ever gene therapy

- 1989 – patient with SCID

- Adenosine Deaminase Deficiency

- AD enzyme needed in purine metabolism

- Worked, but needs continuous treatments

- 2000 –X-linked SCID, 5 of 10 developed leukemia & died

- 2001 – Liver enzyme deficiency, massive inflammatory response, multiple organ failure, death

Candidates for gene therapy

- Criteria:

- 1) Disease causing gene identified ______

- 2) Can clone____/synthesize gene in lab

- 3) Cells for treatment are _______accessible____

- Is the ultimate goal of Translational medicine in a “bench to bedside manner”

- All gene therapy is somatic____ gene therapy

- Currently there is not ____germline____ gene therapy

Approaches for gene delivery

• Two forms:

1) In-vivo

- Gene delivered ___into patient________

2) Ex-vivo

- Cells taken ____out of patient_________

How to deliver genes

- 1. Viral vectors

- (A) Retroviruses

- (B) Adenoviruses

- (C) Lentiviruses

- 2. Non-viral vectors

- (A) Liposomes

- (B) Nanoparticles

- (C) Gene pills

- (D) RNAi

How to deliver genes - viral vectors

- (A) Retroviruses – has an RNA genome

- Process:

- Harmful genes of virus (replication & disease causing) removed

- Cloned human gene inserted into retrovirus

- Infect patient cells

- RNA made into DNA (reverse transcription), inserts into host genome

- Advantages

- (+) = insert into host genome so it is stable__

- (+) = have long term_____ gene expression of correct protein

- (+) = when host cell divides the correct gene is in all new cells______

- Disadvantages

- (-) = can only infect dividing_____ cells

- (-) = can cause toxicity_____ by insertional mutation

- (-) = can ___inactivate_______ essential genes by inserting into them

- (-) = can insert into promoter region causing cancer_____

How to deliver genes - viral vectors

- (B) Adenoviruses

- Process:

- Cloned human gene inserted into adenovirus

- Infect patient cells

- virus delivers genes to nucleus that make an episome_____

- Advantages

- (+) = Can carry medium____ sized genes

- (+) = ___does not_______ inactivate essential genes or cause cancer

- Disadvantages

- (-) = can only infect dividing cells

- (-) = If you have ever been infected by adenovirus immune system will attack_______

- (-) = ____does not______ integrate into host genome so treatment is only ___temporary________

- (-) = when host cell divides, episome is not__ in all new cells

- (-) = patient will need continuous new gene therapy treatments

How to deliver genes - viral vectors

- (C) Lentivirus – a retrovirus (includes HIV)

- Process:

- The disease causing portions of HIV are removed

- Correct gene is inserted into lentivirus

- Infect patient cells

- RNA made into DNA (reverse transcription), inserts into host genome

- Advantages

(+) = Can carry large___ genes

(+) = can__ incorporate into host genome

(+) = can infect ___non diving_________ & dividing cells

(+) = HIV target is T-cells__ so very good for blood disorder

- Disadvantages

- (-) = cannot control where it ___inserts_____, but inserts into essential genes or cancer causing less than retroviruses

How to deliver genes

- 1. Viral vectors

- (A) Retroviruses

- (B) Adenoviruses

- (C) Lentiviruses

- 2. Non-viral vectors

- (A) Liposomes

- (B) Nanoparticles

- (C) Gene pills

- (D) RNAi

How to deliver genes - non-viral vectors

- (A) Liposomes

- Process:

- lipid bilayer__________ vesicle with gene inside

- Fuses with host cell membrane

- Gene endocytosed_________ into cell

- Advantages:

- (+) No size limit_____ of gene to be inserted

- Disadvantages

- (-) does not integrate into host genome

- (-) gene transfer is not as efficient______

- (-) gene expression does not last long

How to deliver genes - nonviral vectors

- (B) Nanoparticles

- Process:

- DNA is a polyanion_____ (due to phosphate group)

- Complexed with polycation______ nanoparticle

- Advantages

- (+) can deliver to dividing & non-dividing cells

- (+) can deliver gene larger than viral delivery methods

How to deliver genes - nonviral vectors

- (C) “gene pills”

- State of the art – only developed in the past 5 years

- A pill delivers the therapeutic DNA

- Pill travels to intestine, DNA is absorbed by cells

- Intestinal cells then express the protein, secrete into blood (D) RNA silencing (RNAi)

- Inhibits mRNA of defective genes

- Can be delivered by plasmid, lentivirus, or inject directly into cell

- RNAi can then bind its complementary mRNA target to inactivate it

- Difficult because RNA is short lived & how to get to healthy vs non-healthy cells

Genetic medicine - gene therapy

- Requirements for approval of clinical trials for gene therapy

- 1. Knowledge of defect/how it causes symptoms

- 2. An animal____ model

- 3. Success in human cells grown in vitro___

- 4. No successful alternative_____ therapies

- 5. safe_ experiments for humans

Gene therapy & Clinical trials

- cancer_____therapies #1 type of clinical trials

- retrovirus_____ #1 type of vector used

- united states______#1 country with clinical trials

- Most clinical trials are in phase 1 ____

Hurdles to gene therapy

- cost___ = treatment is not always permanent & costs $450,000- $1,000,000

- ____single gene only______________= there is not gene therapy for disorders caused by multiple genes

- ethics____ – some gene therapies could breach the Weismann barrier protecting the testes &

affect germline cells

- ____viral vectors________ – can cause toxicity, be attacked by immune cells & inflammatory responses that are dangerous to the patient

- _____not long lasting___________- in some cases the insertion is not stable in the genome & doesn’t integrate properly

- Causes other more _______serious problems__________– if inserted next or within a tumor suppressor gene it could cause cancer

CRISPR-cas 9

- Can now edit parts of the genome by removing, adding, or altering sections of DNA

- Naturally occurs in bacteria to repair viral damage

- Made of 2 molecules

- 1) cas 9_____ enzyme = “molecular scissors” cut DNA in a very specific place

- 2) ___gRNA____ = “guide RNA” is predesigned 20 bases long RNA that guides the Cas9 to the correct place. Is complementary to the DNA that will be cut out

- Advantage = cheap, fast, good results

- Disadvantage = off-target binding/cutting (what if 19 of 20 bases match?)

Stem cells

- All cells arise from the ultimate stem cell = ___fertilized egg___________

- Zygote – first few cell divisions are symmetric = all daughter cells have the same ___potency______ (potential for developing into different cell types)

- After this point cells become :

- 1) committed_______ to pathways to become different cell types &

- 2) ____restricted _______ in their capacity to generate different types of cells

- when a stem cells divides one cell remains a stem cell and the other one differentiates

Stem cell therapy

- allogenic_____ cell therapy = transplanted cells come from ___a donor______

- are genetically different from those cells of the recipient

- high risk of immune rejection, must HLA match

- autologous________ cell therapy = transplanted cells come from themselves________

- avoids immune rejection

- typically involves reprogramming nuclei & gene expression

iPS

- Induced pluripotent stem cells (iPS) = adult cells (ex. skin cells) that are reprogrammed to become late stage_________ embryonic stem cells.

- + can make pluripotent cells that are specific to a _____disease______

- + allows the study the pathway____ of particular disease

- + allows testing ___treatments________ to cure that disease

- + are from the patient so minimal risk of rejection____

- Mesenchymal stem cells = found throughout body

- Can direct to become bone, cartilage, fat, muscle

What can be treated successfully right now

- Currently approved stem cell treatments

- 1) Bone marrow transplants (50 years) – hematopoietic stem cells

- Used to treat blood cancers (leukemia, lymphoma, sickle cell, etc)

- Needle placed into soft center of donor bone marrow or collecting peripheral

blood stem cells by ____ampheresis________ →Collected stem cells transfused into recipient

2) _____umbilical cord____________blood (1988) – hematopoietic stem cells

- Blood that remains in the placenta/umbilical cord after childbirth

- Cannula used to remove blood, stem cells isolated, cryopreserved until needed Stem cell treatments that are the furthest along in clinical trials

• Bone

• Skin

• Cornea

What to warn your patient about

- ____clinical treatment_____________= medical practice that has been shown through formal

clinical trials to be reasonably safe & effective for treating a particular

disease/condition & approved through FDA

- clinical trial_______ = research study to answer specific questions about a new

treatment. Seeks to establish if new treatments are safe & effective. The

treatment is thus far unproven.

- ____expermential intervention__________________ = new, untested, or different from usual medical treatment. Has not been proven that is safe or if it will work in treating disease