Gene Therapy

1
Stem cells and Gene Therapy
Revision of
BPH-307 PHARMACOLOGY III
Unit 5
by

• Abhijit Debnath

Asst. Professor PDM University

• Website Theabhi.in

• E-mail abhijit.debnath_at_pdm.ac.in

2
Syllabus
3
Syllabus
Gene Therapy
Stem Cells

1. History of Gene Therapy
2. Introduction to gene therapy
3. Types
4. Methods
5. Applications of gene therapy.

• History stem cells
• Introduction to stem cells
• types of stem Cells
• Human stem cell culture techniques
• Applications of stem cells
• Ehhical issues related to stem cells

4

1. History of Gene Therapy

5
1. History of Gene Therapy
In 1972 Friedmann and Roblin authored a paper in
Science titled "Gene therapy for human genetic
disease? Rogers (1970) was cited for proposing
that exogenous good DNA be used to replace the
defective DNA in those who suffer from genetic
defects.
1970s earlier
In 1984 a retrovirus vector system was designed
that could efficiently insert foreign genes into
mammalian chromosomes.
1980s

• The first approved gene therapy clinical research
  in the US took place on 14 September 1990, at the
  National Institutes of Health (NIH) USA
• Cancer gene therapy was introduced in 1992/93 .
• In 1992, first gene therapy procedure using
  hematopoietic stem cells as vectors to deliver
  genes intended to correct hereditary diseases.

1990s

• The first approved gene therapy clinical research
  in the US took place on 14 September 1990, at the
  National Institutes of Health (NIH) USA
• Cancer gene therapy was introduced in 1992/93 .
• In 1992, first gene therapy procedure using
  hematopoietic stem cells as vectors to deliver
  genes intended to correct hereditary diseases.

1990s
6
1. History of Gene Therapy
The modified cancer gene therapy strategy of
antisense IGF-I RNA (NIH n 1602) using antisense
/ triple helix anti-IGF-I approach was registered
in 2002 by Wiley gene therapy clinical trial - n
635 and 636.
2000s

• Sickle-cell disease can be treated in mice
• A new gene therapy approach repaired errors in
  messenger RNA derived from defective genes. This
  technique has the potential to treat
  thalassaemia, cystic fibrosis and some cancers.
• Researchers created liposomes 25 nanometers
  across that can carry therapeutic DNA through
  pores in the nuclear membrane

2002

• In 2003 a research team inserted genes into the
  brain for the first time. They used liposomes
  coated in a polymer called polyethylene glycol,
  which unlike viral vectors, are small enough to
  cross the bloodbrain barrier.
• Short pieces of double-stranded RNA (short,
  interfering RNAs or siRNAs) are used by cells to
  degrade RNA of a particular sequence. If a siRNA
  is designed to match the RNA copied from a faulty
  gene, then the abnormal protein product of that
  gene will not be produced.
• Gendicine is a cancer gene therapy that delivers
  the tumor suppressor gene p53 using an engineered
  adenovirus.
• In China for the treatment of head and neck
  squamous cell carcinoma was approved.

2003
7
1. History of Gene Therapy

• In March, researchers announced the successful
  use of gene therapy to treat two adult patients
  for X-linked chronic granulomatous disease, a
  disease which affects myeloid cells and damages
  the immune system. The study is the first to show
  that gene therapy can treat the myeloid system.
• In August, scientists successfully treated
  metastatic melanoma in two patients using killer
  T cells genetically retargeted to attack the
  cancer cells.
• In November, researchers reported on the use of
  VRX496, a gene-based immunotherapy for the
  treatment of HIV that uses a lentiviral vector to
  deliver an antisense gene against the HIV
  envelope.

2006

• In May, researchers announced the first gene
  therapy trial for inherited retinal disease. The
  first operation was carried out on a 23-year-old
  British male, Robert Johnson, in early 2007.

2007

• Delivery of recombinant adeno-associated virus
  (AAV) carrying RPE65 yielded positive results in
  Leber's congenital amaurosis is an inherited
  blinding disease caused by mutations in the RPE65
  gene.

2008

• In September, researchers were able to give
  trichromatic vision to squirrel monkeys.
• In November, researchers halted a fatal genetic
  disorder called adrenoleukodystrophy in two
  children using a lentivirus vector to deliver a
  functioning version of ABCD1, the gene that is
  mutated in the disorder.

2009
8
1. History of Gene Therapy
2010s

• An April, paper reported that gene therapy
  addressed achromatopsia (color blindness) in dogs
  by targeting cone photoreceptors. Cone function
  and day vision were restored for at least 33
  months in two young specimens. The therapy was
  less efficient for older dogs.
• In September, it was announced that an
  18-year-old male patient in France with
  beta-thalassemia major had been successfully
  treated
• Cancer Immunogene therapy using modified
  antigene, antisense/triple helix approach was
  introduced and gene therapy targeting IGF-I, the
  IGF-I expressing tumors i.e. lung and epidermis
  cancers were treated

2010

• In 2007 and 2008, a man (Timothy Ray Brown) was
  cured of HIV by repeated hematopoietic stem cell
  transplantation with double-delta-32 mutation
  which disables the CCR5 receptor. This cure was
  accepted by the medical community in 2011. It
  required complete ablation of existing bone
  marrow, which is very debilitating.
• In August, two of three subjects of a pilot study
  were confirmed to have been cured from chronic
  lymphocytic leukemia (CLL).
• Neovasculgen was registered in Russia as the
  first-in-class gene-therapy drug for treatment of
  peripheral artery disease, including critical
  limb ischemia

2011
9
1. History of Gene Therapy

• In July, the FDA approved Phase 1 clinical trials
  on thalassemia major patients in the US for 10
  participants.
• In July, the European Medicines Agency
  recommended approval of a gene therapy treatment
  for the first time in either Europe or the United
  States. The treatment used Alipogene tiparvovec
  (Glybera) to compensate for lipoprotein lipase
  deficiency, which can cause severe pancreatitis.

2012

• In March, researchers reported that three of five
  adult subjects who had acute lymphocytic leukemia
  (ALL) had been in remission for five months to
  two years after being treated with genetically
  modified T cells which attacked cells with CD19
  genes on their surface, i.e. all B-cells,
  cancerous or not.
• In July, researchers reported promising results
  for six children with two severe hereditary
  diseases had been treated with a partially
  deactivated lentivirus to replace a faulty gene
  and after 732 months. Three of the children had
  metachromatic leukodystrophy, which causes
  children to lose cognitive and motor skills.
• In October, researchers reported that two
  children born with adenosine deaminase severe
  combined immunodeficiency disease (ADA-SCID) had
  been treated with genetically engineered stem
  cells 18 months previously and that their immune
  systems were showing signs of full recovery.
• In October, researchers reported that they had
  treated six hemophilia sufferers in early 2011
  using an adeno-associated virus.

2013
10
1. History of Gene Therapy

• In January, researchers reported that six
  inherited genetic eye disease (choroideremia)
  patients had been treated with adeno-associated
  virus with a copy of REP1. Over a six-month to
  two-year period all had improved their sight.
• In March, researchers reported that 12 HIV
  patients had been treated since 2009 in a trial
  with a genetically engineered virus with a rare
  mutation (CCR5 deficiency) known to protect
  against HIV with promising results.

2014

• In February, LentiGlobin BB305, a gene therapy
  treatment undergoing clinical trials for
  treatment of beta thalassemia gained FDA
  "breakthrough" status after several patients were
  able to forgo the frequent blood transfusions
  usually required to treat the disease.
• In March, researchers delivered a recombinant
  gene encoding a broadly neutralizing antibody
  into monkeys infected with simian HIV the
  monkeys' cells produced the antibody, which
  cleared them of HIV.
• In March, scientists, including an inventor of
  CRISPR, Jennifer Doudna, urged a worldwide
  moratorium on germline gene therapy, writing
  "scientists should avoid even attempting, in lax
  jurisdictions, germline genome modification for
  clinical application in humans" until the full
  implications "are discussed among scientific and
  governmental organizations".
• In October, researchers announced that they had
  treated a baby girl, Layla Richards, with an
  experimental treatment using donor T-cells
  genetically engineered using TALEN to attack
  cancer cells.
• In December, scientists of major world academies
  called for a moratorium on inheritable human
  genome edits, including those related to
  CRISPR-Cas9 technologies but that basic research
  including embryo gene editing should continue.

2015
11
1. History of Gene Therapy

• In April, the Committee for Medicinal Products
  for Human Use of the European Medicines Agency
  endorsed a gene therapy treatment called
  Strimvelis and the European Commission approved
  it in June. This treats children born with
  adenosine deaminase deficiency and who have no
  functioning immune system. This was the second
  gene therapy treatment to be approved in Europe.
• In October, Chinese scientists reported they had
  started a trial to genetically modify T-cells
  from 10 adult patients with lung cancer and
  reinject the modified T-cells back into their
  bodies to attack the cancer cells. The T-cells
  had the PD-1 protein (which stops or slows the
  immune response) removed using CRISPR-Cas9.

2016

• In February, Kite Pharma announced results from a
  clinical trial of CAR-T cells in around a hundred
  people with advanced Non-Hodgkin lymphoma.
• In March, French scientists reported on clinical
  research of gene therapy to treat sickle-cell
  disease.
• In August, the FDA approved tisagenlecleucel for
  Acute lymphoblastic leukemia
• In December, the results of using an
  adeno-associated virus with blood clotting factor
  VIII to treat nine haemophilia A patients were
  published.
• In December, the FDA approved Luxturna, the first
  in vivo gene therapy, for the treatment of
  blindness due to Leber's congenital amaurosis.The
  price of this treatment was 850,000 US dollars
  for both eyes.

2017
12
1. History of Gene Therapy

• In March, a New way to potentially fight Ebola
  was reported
• In April, CRISPR/Cas9 silences gene associated
  with high cholesterol
• In June, Genome-editing tool could increase
  cancer risk
• In October, Preventing sudden cardiac death with
  genome editing
• In November, New study offers hope for patients
  suffering from a rare form of blindness
• In November, Realizing the potential of gene
  therapy for neurological disorders like
  sclerosis and Parkinson's disease

2018

• In February 2019, medical scientists working of
  California, announced the first ever "in body"
  human gene editing therapy to permanently alter
  DNA - in a patient with Hunter Syndrome. Clinical
  trials by Sangamo involving gene editing using
  Zinc Finger Nuclease (ZFN) are ongoing.

2019
13

• 2. Introduction to gene therapy

14
What is the Most Common thing in all these
diseases???

• Gene therapy is an experimental technique that
  uses Genes to treat or prevent disease.

• The First Success Story of Gene Therapy
• The first human to receive gene therapy treatment
  was a 4 year old girl with severe
  immune-deficiency disease.
• This disease is caused by a faulty gene that
  fails to produce a vital enzyme.
• In the therapy procedure, they extracted some of
  the girl's white blood cells.
• Then, they exposed them to a genetically
  engineered virus that had lost its virulence but
  still carried normal versions of the gene that
  was not functioning correctly in the girl.
• The virus invaded the white blood cells, and then
  these cells were transfused back into the girl.
• Once back inside the girl's bloodstream, the
  cells began producing the proper enzyme.
• Although the girl still needs follow-up
  treatments, she now leads a relatively normal
  life following the gene therapy.
• This is one of the success stories of gene
  therapy.

• In this to treat a disorder a gene is inserted
  into a patient's cells instead of using drugs or
  surgery.

• The first attempt at modifying human DNA was
  performed in 1980 by Martin Cline

• But the first successful nuclear gene transfer in
  humans was approved by the National Institutes of
  Health, was performed in May 1989.

Gene Therapy

• The first therapeutic use of gene transfer as
  well as the first direct insertion of human DNA
  into the nuclear genome was performed by French
  Anderson in a trial starting in September 1990.

• Between 1989 and February 2016, over 2,300
  clinical trials were conducted, with more than
  half of them in phase I

15

• 3. Types of Gene Therapy

16
1. Types of Gene Therapy
17

• 4. Methods of Gene Therapy

18
4. Methods of Gene Therapy
19

• 5. Applications of gene therapy

20
5. Applications of gene therapy
1. Non-Viral Delivery Systems in Gene Therapy 2.
Plasmid Transgene Expression in vivo 3. Silencing
of Transgene Expression A Gene Therapy
Perspective 4. Cellular Uptake Mechanism of
Non-Viral Gene Delivery and Means for Improving
Transfection Efficiency 5. Polylipid
Nanoparticle, a Novel Lipid-Based Vector for
Liver Gene Transfer 6. DNA Electrotransfer An
Effective Tool for Gene Therapy 7. siRNA and Gene
Formulation for Efficient Gene Therapy 8.
Mesenchymal Stem Cells as Gene Delivery
Vehicles 9. Cancer Gene Therapy Key Biological
Concepts in the Design of Multifunctional
Non-Viral Delivery Systems 10. Gene Therapy
Based on Fragment C of Tetanus Toxin in ALS A
Promising Neuroprotective Strategy for the Bench
to the Bedside Approach 11. Transposons for
Non-Viral Gene Transfer 12. Lentiviral Gene
Therapy Vectors Challenges and Future Directions
21
5. Applications of gene therapy
13. Lentiviral Vectors in Immunotherapy 14.
Targeted Lentiviral Vectors Current Applications
and Future Potential 15. Vectors for Highly
Efficient and Neuron-Specific Retrograde Gene
Transfer for Gene Therapy of Neurological
Diseases 16. Retroviral Genotoxicity 17.
Efficient AAV Vector Production System Towards
Gene Therapy For Duchenne Muscular Dystrophy 18.
Gene Therapy for Primary Immunodeficiencies 19.
Gene Therapy for Diabetic Retinopathy Targeting
the Renin-Angiotensin System 20. Gene Therapy
for Retinitis Pigmentosa 21. Gene Therapy for
Erythroid Metabolic Inherited Diseases 22.
Targeting the Lung Challenges in Gene Therapy
for Cystic Fibrosis 23. Gene Therapy for the
COL7A1 Gene 24. Molecular Therapy for Lysosomal
Storage Diseases 25. Gene Therapy Perspectives
Against Diseases of the Respiratory System
22
5. Applications of gene therapy
26. Gene Therapy in Critical Care Medicine 27.
Clinical and Translational Challenges in Gene
Therapy of Cardiovascular Diseases 28. Gene
Therapy for Chronic Pain Management 29. Insulin
Trafficking in a Glucose Responsive Engineered
Human Liver Cell Line is Regulated by the
Interaction of ATP-Sensitive Potassium Channels
and Voltage-Gated Calcium Channels 30.
Feasibility of Gene Therapy for Tooth
Regeneration by Stimulation of a Third Dentition