This document discusses various topics related to gene therapy including its applications in cancer, HIV, and hemophilia. It discusses CAR receptor technology, the HACE lipoplex method, CRISPR/Cas9, mRNA vaccines, and the immune response to gene therapy. It also summarizes the case of Jesse Gelsinger who died in a gene therapy trial for a rare metabolic disorder and methods of gene knockout.

1. ADD ON POINTS OF GENE
THERAPY SEMINAR

2. QUESTIONS???
• Gene therapy in Cancer, HIV, Hemophilia, Heart disease
• CAR Receptor
• HACE lipoplex
• Levo dopa and Carbi dopa mechanism of action
• Immune response to gene therapy
• Jesse Gelsinger case
• Gene Knockout
• CRISPR/Cas9
• mRNA vaccine – gene therapy or not

3. 1. GENE THERAPY
A. CANCER
(Cross D, Burmester JK. Gene therapy for cancer treatment: past, present and future. Clin Med
Res. 2006;4(3):218-227. doi:10.3121/cmr.4.3.218)

4. Contd…
• CAR19 T cells were used for the treatment of an infant with refractory relapsed acute B-
lymphoblastic leukemia (B-ALL) with great success
(Qasim WAS,SamarasingheS First clinical application of Talen engineered universal CAR19 T cells in B-ALL Blood
2015;126 (23):2046)
• In hepatocellular carcinoma (HCC), recently it has been demonstrated that fibroblast growth
factor receptor 4 (FGFR4) knockout by CRISPR technology can make HCC cancer cells sensitized to
sorafenib
(Ardelt MAFT Martini E Inhibition of Cyclin-dependent kinase 5 – a novel strategy to improve Sorafenib
response in HCC therapy Hepatology 2019 ;69 :376)

5. B. HIV
• VRX496, a gene-based immunotherapy for the treatment of HIV that uses a lentiviral vector to
deliver an antisense gene against the HIV envelope in phase I clinical trial, chronic HIV infection
who had failed to respond to at least two antiretroviral regimens were treated.
(Levine BL, Humeau LM, Boyer J, MacGregor RR, Rebello T, Lu X, et al. (November 2006). "Gene transfer in humans using a conditionally replicating
lentiviral vector". Proceedings of the National Academy of Sciences of the United States of America. 103 (46): 17372–17377.)
• In 2007 and 2008, a man (Timothy Ray Brown) was cured of HIV by repeated hematopoietic stem
cell transplantation (see also allogeneic stem cell transplantation, allogeneic bone marrow
transplantation, allotransplantation) with double-delta-32 mutation which disables
the CCR5 receptor. This cure was accepted by the medical community in 2011.
( Rosenberg T (27 May 2011). "The Man Who Was Cured of HIV and What It Means for a Cure for AIDS". New York. Retrieved 2 January 2022.)

6. GENE THERAPY IN HEMOPHILIA
• Hemophilia A and B are congenital bleeding disorders caused by a dysfunction or deficiency of
coagulation factor (F) VIII or IX, respectively
• Genetically modified adeno - associated virus (AAV) engineered to deliver either the FVIII or FIX
gene to the liver, leading to the continuous endogenous synthesis and secretion of the missing
coagulation factor and preventing or reducing bleeding episodes.
(Amit C. Nathwani; Gene therapy for hemophilia. Hematology Am Soc Hematol Educ Program 2022;
2022 (1): 569–578.)

7. CURRENT STUDIES GOING FOR CARDIOVASCULAR
DISEASES

8. 2. CAR RECEPTOR(COXSACKIEVIRUS AND
ADENOVIRUS RECEPTOR RECEPTOR)
• Membrane receptor for coxsackie
viruses and adenoviruses.
• Expressed in several tissues,
including heart, brain, and, more
generally, epithelial and endothelial cells
• Involved in cell adhesion, immune cell
activation, synaptic transmission, and
signaling
(Abdolazimi, Y., Mojarrad, M., Pedram, M., & Modarressi, M. H. (2007). Analysis of the expression of coxsackievirus and
adenovirus receptor in five colon cancer cell lines. World journal of gastroenterology, 13(47), 6365–6369.
https://doi.org/10.3748/wjg.v13.i47.6365)

9. 3. CASE OF JESSE GELSINGER
• Jesse had a rare metabolic disorder called ornithine transcarbamylase deficiency syndrome, or
OTCD, in which ammonia builds up to lethal levels in the blood.
• Babies born with OTCD usually fall into comas soon after birth and suffer brain damage. die
within a month.
• Jesse’s milder version of the deficiency was diagnosed when he was two years old
• In 1999, Clinical trial for a potential OTCD treatment was in the works at the University of
Pennsylvania in Philadelphia .Patients would be injected with working copies of the gene that had
been attached to an adenovirus.
• The virus, altered to be harmless, would infect the patients’ liver cells and integrate the added
gene into their chromosomal DNA.
• Within a day he became disoriented and showed signs of jaundice. He had an intense
inflammatory response and developed a dangerous blood-clotting disorder, followed by kidney,
liver, and lung failure.
• Four days after receiving the shot Jesse was declared brain dead and taken off life support.

10. 4. IMMUNE RESPONSE TO GENE THERAPY
• Innate immune response : early, not antigen specific, and no immunological memory.
• Adaptive immune response: rely on activation and clonal expansion of antigen-specific (effector) B
and T cell differentiation, and generate immunological memory
• Recognition of viral structures (e.g., capsids or nucleic acids) produces immune response

11. IMMUNE RESPONSE MECHANISM AGAINST
AAV VECTORS

12. 5. Lipoplex (HACE)
HACE-based lipo-somes were prepared using cationic
lipid DOTAP and fusogenic helper lipid DOPE .
Then, cationic lipo-somes were prepared placing HA on
the surface and CE buried in the lipid bilayer
*1,2-dioleoyl-3-trimethylammonium-propane
(DOTAP) and 1,2-dioleoyl-sn-glycero-3-
phoshphoethanola-
mine (DOPE) lipids

13. 6. CRISPR cas9
• Genome editing tool
• consists of two key molecules
• Cas9- enzyme act as molecular scissors
• gRNA- pre-designed RNA sequence (about 20
bases long) located within a longer RNA
scaffold
• The scaffold part binds to DNA and the pre-
designed sequence ‘guides’ Cas9 to the right
part of the genome
• Cas9 follows the guide RNA to the same
location in the DNA sequence and makes a cut
across both strands of the DNA.
• Cell recognises that the DNA is damaged and
tries to repair it.
(Clustered Regularly Interspaced Short Palindromic
Repeats)

14. 7. LEVO DOPA AND CARBI DOPA IN
PARKINSONISM
• Levodopa is converted to dopamine via the action of a naturally
occurring enzyme called DOPA decarboxylase.
• This occurs both in the peripheral circulation and in the central nervous system after
levodopa has crossed the blood brain barrier.
• Usually administered in combination with a DOPA decarboxylase inhibitor (DDCI),
(carbidopa), which is very polar (and charged at physiologic pH) and cannot cross the
blood brain barrier, prevents peripheral conversion of levodopa to dopamine
• Reduces peripheral side effects of levodopa. (Nausea, vomiting)
• Carbidopa also increases the quantity of levodopa in the bloodstream that is available to
enter the brain.

15. 8. GENE KNOCKOUT
• a processof gene inactivation or
removal
• types: complete and conditional
• Methods for gene knockout are
enlisted here:
1.Gene silencing
2.Conditional knockout
3.Homologous recombination
4.Gene editing
5.Knockout by mutation

16. 9. mRNA VACCINES
• An mRNA vaccine is a type of vaccine that uses a copy of a molecule called messenger
RNA (mRNA) to produce an immune response.
• The vaccine delivers molecules of antigen-encoding mRNA into immune cells, which use the
designed mRNA as a blueprint to build foreign protein that would normally be produced by
a pathogen (such as a virus) or by a cancer cell.
• These protein molecules stimulate an adaptive immune response that teaches the body to
identify and destroy the corresponding pathogen or cancer cells

17. Mechanism

18. Contd…
• As mRNA is genetic material, mRNA vaccines can be looked at as a genetic-based therapy, but
they are classified as vaccines and are not designed to alter your genes
• Gene therapy, in the classical sense, involves making deliberate changes to a patient’s DNA in
order to treat or cure them. mRNA vaccines will not enter a cell’s nucleus that houses your DNA
genome. There is zero risk of these vaccines integrating into our own genome or altering our
genetic makeup.
Quoted by Dr Adam Taylor, a virologist and research fellow at the Menzies Health Institute,
Queensland, Griffith University