Gene Therapy

Dose escalation study

- each person gets a little bit more before- to find out dosages for side

Somatic Cell

All body cells except for gametes (egg and sperm).

Gene Therapy

Any procedure intended to treat or alleviate disease by genetically modifying the cells of a patient. -2 types: somatic of germ line

Was ADA SCID gene therapy trial ex vivo or in vivo? Was it somatic cell gene therapy or germ line gene therapy?

Ex Vivo, Somatic Cell

Leber's Congenital Amaurosis (LCA)

LCA is a group of congenital recessive disorders leading to blindness, one of the most severe inherited form of retinal disorders. The retina is the specialized tissue at the back of the eye that detects light and color. Leads to severe visual impairment in childhood, total blindness in adulthood. At least 13 types have been described and are distinguished by genetic cause, patterns of vision loss, and related eye abnormalities. -currently there is no treatment

Cavazzana-Calva Study 2000

SCID X1 • Removed bone marrow cells from 2 patients • Transduced using retroviral vector with working copy of yc gene • Put cells expressing yc back in patient • 10 months later both boys still had immune function

Vector

a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it could be replicated or expressed.

Maguire 2009 Phase 1 Trial

can we get the gene in w/o causing harm • 12 patients age 8-44, dose escalation study using the adeno-associated viral vector containing a gene for human RPE65 cDNA o no serious ramifications • all 12 individuals reported improved vision starting just 2 weeks after surgery, substantial and stable at all 3 doses • earlier patients are injected- the more effective the treatment is o Testa 2013 3 year follow up • Still no adverse events • Maximal improvement at 6 months and then stayed stable

Jesse Gelsigner

died 4 days after injection of the vector (OTC Dose Esc. Study) • the vector initiated a systemic immune response that led to multiple organ failure and death • 2000 Gelsinger family settled lawsuit • Jan. 2001 FDA halted gene therapy and other clinical trials at U of Penn • March 2005 U Penn and Children's National Center paid $1 million to settle fraud allegations

Ex-Vivo Gene therapy "in vitro"

o Cells are removed from the patient o Genetic material is inserted into them o Then transplant the modified cells back into the patient o Considered to be safer o Limitation: cells normally don't like to be taken out and put back in o Works with bone marrow, skin, and blood cells easy to culture and extract -somatic

In-Vivo Gene therapy

o Genetic material is transferred directly into cells within a patient o May be the only kind of gene therapy if it's a cell that you can't take out and put back in (ex. brain, lungs) -somatic

SCID X1

o Mutation on the X chromosome, x-linked recessive form of SCID involving the gene encoding the γc cytokine receptor. Immune system gene does not to grow, boys

Study of LCA2

o Particular study study of LCA2 is due to mutations in RPE65 (gene provides instructions for making a protein that is essential for normal vision, provides a thin layer of cells at the back of the eye, which supports and nourishes the retina, which is light sensitive tissue that lines the back of the eye. . Accounts for 6% Leber cases

Promising Treatment

o Progeny (descendants) of HSCs (hematopoietic stem cells) that were engineered to carry the correct version of a gene (through the integration of a lentiviral vector) distribute thru the body. Cartier show that some cells replaced diseased microglia in the brain and relieved lipid storage in patients suffering from ALD

Adrenoleukodystrophy

o X-linked (boys) ALD is a fatal demyelinating disease caused by mutations in ABCD1 encoding the ALD protein, neurons lose insulation(the myelin protective sheath that surrounds neurons) • Symptoms around 6 yrs old, brain stops functioning • Bone marrow works • Has to be done early because you can't go backwards with brain

Maguire Experiment Steps

o use a vector and put a working gene in that vector, injected the eye • Injected in eye with the worst function with the vector, and in the other just a solution (in vitro) • Each patient was the experiment in one eye, and one eye was the control • 3 different doses (groups): low medium and high

2003, SCIDX1, where does the vector go in the cells?

o virus inserts the DNA into the cells DNA o inserted the DNA in the cells right next to or into an oncogene (cancer causing gene) (LMO2) implicated in childhood leukemia o promoter that they put with the gene in the vector said make a lot of copies of gene, and got next to oncogene, that gene goes crazy

Cartier 2009

used lentiviral vector instead (HIV1 derived) with ABCD1 gene o Two 7 yr old boys who had progressive ALD and no matched donor, were treated o 24-30 month follow up indicated progression of the disease had been halted

Human Gene Therapy Trials

• - review still happens at institutional and FDA level, NIH not approval necessarily anymore

UPenn OTC Dose Escalation Study

• 18 subjects: clinically stable adults • vector: modified human adenovirus • Virus was injected into the hepatic vein (leads to the liver) • All subjects had a clinical response to the injection (ex. Fever) and a few also had liver inflammation (but it self corrected within 2 weeks. • 18th Subject, Jesse Gelsigner

Other Somatic Cell Gene therapy trials are for...

• Hemophilia • Muscular dystrophy • Cystic fibrosis • Heart disease • Arthritis

Severe combined Immunodeficiency (SCID)

• Immune system is nonfunctional, leaving them vulnerable to infection. Problem is a mutant gene that prevents production of an enzyme called adenosine deaminase (ADA). W/o ADA, white blood cells never mature, they die while still developing in the bone marrow. - patient has neither cell mediated immune response nor the ability to make antibodies • 25% of SCID cases are due to a recessive mutation in the ADA gene

Different SCID Treatments

• Keep away from the environment in a bubble - David o David had bone marrow from sister, who had mono in the past, and killed him • Give the person with SCID an immune system, thru bone marrow o Match so you don't want the donor cells to attack the recipient • 50% cure rate • Injection of the enzyme itself (ADA) from cows, every week

o Somatic Cell Gene therapy limits

• Kinds of diseases that are treatable • So far, diseases that are due to a single gene, and that are recessive o When a gene is recessive, it's due to the lack of a gene product o When dominant, it's not due to a lack of a gene product, it's due to having the wrong gene product, we'd have to get rid of the wrong one and introduce a new one • Problems with Vectors • Vector can be the problem, need to find safer more effective vectors • Expensive • Research for rare? Life saving? Limited health care resources • Treatment for one Individual (in the body) • Not generational, they will still pass it on.

SCID experiment

• Little girl Ashanti de Silva- 1990 -receives treatment in the form of a gene • White blood cells were genetically engineered to carry the ada gene and then return to her body • First gene therapy for humans

Disturbing Details of OTC Study

• Mutations in vector • Vector doesn't go just to the liver • Did not report adverse affects • Choice of Subjects • Financial conflict of interest

Germ Line Gene Therapy

• Risk to future generations, not just the individual, it will be passed on if you reproduce • Can you give informed consent for your children, grandchildren, great grandchildren • Should you be able to? • Reduction of genetic diversity • Gene's we find deleterious at this time we consider this way because of our environment, it might be different in the future. • Not necessary • We can use in vitro • Current technology not ready

Results of SCID treatment

• T cells don't live that long, lasted 6 months • 2002 bone marrow instead of blood - that was stable o Follow up paper 2009, 10 children, all patients still alive. • 8 of 10 children have stable working immune systems • 2 are still getting the injections

SCID Protocol Treatment

• Take out white blood cell, put gene into white blood cell in a dish, using a vector (carrier molecule) known as retroviral, virus gets gene into cell, mouse leukemia virus • Transfect the ADA vector into cultured T lymphocytes from patient • Identify the ADA+ T cells and expand them in culture • Reimplant cells in patient

Hacein-Bey-Abina Follow up study 2002

• Treat additional 5 boys, with ex vivo gene therapy • April 4 of 5 appeared to have normal working immune systems • October 2002, little boy develops leukemia • Jan 2003 a 2nd boy develops leukemia • Fall 2004 one child died • Jan 2005 3rd case reported, then 4th • May 2005 US advisory panel decided that when 2 X-SCID trials in US resume they should only treat children for whom bone marrow

Somatic Cell or Non-heritable Gene Therapy

• Treating the gene in somatic cells • Treat a disease symptoms or hopes to • Not a cure in a heritable sense -2 types: in vivo or ex vivio

Genetic Enhancement

• Use of genetic engineering to supply a characteristic that a parent might want in a child that does not involve the treatment or prevention of a disease (non disease phenotype)

FDA Center for Biologics Evaluation and Research

• Uses the same protocol as investigating a drug -everything is at the experimental level right now • Gene that they want to insert needs to be well characterized and cloneable • Need an effective delivery method of cell • Disease must not be treatable by anything else • Does not create more health risks than the individual's life already would be • Must be preliminary data - research done on cells, and often animal model systems, demonstrating the gene therapy is effective not harmful • 3 phase clinical trial process 1. Small Pilot (6-10 people) to test safety 2. Larger number of people, is it effective? 3. Larger sample aimed at measuring long term safety

FDA Violations of Dose Escalation Study

• Why didn't you tell us the first 17 had any adverse effects? • Penn says but reactions weren't so good • 2 animal trials, 1 died, • Penn- not comparable, the animals got so much more of the vector and it was a different vector • Galinger's blood levels of ammonia levels were too high

Germ-Line or heritable gene therapy

• Will be passed on • Change the genes in the germ line, which are your reproductive cells • Not currently being done in humans • Might be cure able

Ornithine Transcarbamylace (OTC) deficiency

• X linked recessive affected individuals cannot process nitrogen properly leading to an accumulation of ammonia in the blood, can't process nitrogen, fatal at infancy • OTC gene has been isolated and characterized • Restoration of enzyme activity in the liver is expected to normalize metabolism (liver transplants can be an effective treatment) • Able to control with drugs the ammonia • Women who are carriers (heterozygous) show mild symptoms