2. Outline
Introduction to gene therapy
Global gene therapy market
Significance in therapeutics
Success of gene therapy
Current scenario
Challenges
3. ―It's a history book - a narrative of the journey of our
species through time. It's a shop manual, with an
incredibly detailed blueprint for building every
human cell.
It's a transformative textbook of medicine, with
insights that will give health care providers immense
new powers to treat, prevent and cure disease."
- Francis Collins
Introduction to Gene Therapy
I. Genome = approx. 3 billion base pairs
II. One base pair is 0.00000000034 meters
III. DNA sequence in any two people is 99.9% identical – only
0.1% is unique!
4. Genetic disorders
Single gene disorders caused by a mistake in
a single gene. Sickle cell, cystic fibrosis and
Tay-Sachs
Chromosome disorders caused by an excess
or deficiency of the genes. Down syndrome is
caused by an extra copy of a chromosome
(but no individual gene on the chromosome is
abnormal)
Multifactorial inheritance disorders caused by
a combination of small variations in genes,
most cancers, Alzheimer's.
5. Gene therapy
In 1865 Mendel’s observations led to laws regarding
the transmission of hereditary characteristics from
generation to generation
1940s - Avery and colleagues
1953 –Watson and Crick
1990 - The first gene therapy journal published,
Human Gene Therapy
1990 - Ashanthi DeSilva, ADA-deficient Severe
Combined Immunodeficiency
2000 - The first cure Alain Fischer (Paris) totally
correcting children with SCID-X1
7. The once abandoned gene therapy field has become a hotbed, with 11
different companies raising at least $618 million from venture capitalists
and the public markets since the beginning of 2013, and one more, AGTC
(Australasian Gene Therapy Society) plans a $50 million initial
public offering soon. The iShares Nasdaq Biotechnology Index is up 65%
in 12 months.
In March 2014 researchers at the University of Pennsylvania reported that
12 patients with HIV had been treated since 2009 in a trial with a
genetically engineered virus with a rare mutation known to protect against
HIV (CCR5 deficiency) top talent is being attracted to what was once seen
as a lost cause
Cedars-Sinai Regenerative Medicine Institute has received a $2.5 million
grant from the Department of Defense to conduct animal studies that, if
successful, could provide the basis for a clinical trial of a gene
therapy product for patients with Lou Gehrig's disease, also called
amyotrophic lateral sclerosis(ability to initiate and control muscle
movement is lost.)-
News medical, BBC 31.3.13
9. Means of gene therapy
Viruses- adenoviruses, retro viruses (unlike Ad, transfect by integrating the transgene
into the target-cell chromosome)
Naked gene transfer- to elucidate mechanisms of gene expression and the
role of genes and their cognate proteins in the pathogenesis of disease in
animal models also being used in several human clinical trials like- genetic
vaccines, Duchenne muscular dystrophy, peripheral limb ischemia, cardiac
ischemia
Needle-free injection- gene gun, Intraject or Jetgun
Electroporation - involves the application of a pulsed electric field enhance
cell permeability, resulting in the transit of exogenous polynucleotide
across the cytoplasmic membrane performed by locally injecting DNA to
the site of interest followed by the application of electric field
local injection < systemic injection
Condensed DNA particles- polymers are heterogeneous polylysine,
polyethylene imine
Liposomes
Burdette, Walter J. The Basis for Gene Therapy. Springfield: 2001
11. Success of gene therapy
GT drugs as Anti-cancer
The increasing popularity of cancer therapeutics as a major interest for
gene therapy applications led to it accounting for a dominant share of more
than 60% in the overall number of clinical studies
Rexin-G A tumor-targeted retrovector bearing a cytocidal cyclin G1 construct,
is the first targeted gene therapy vector to gain fast track designation and
orphan drug priorities for multiple cancer indications in US
Gendicin Recombinant Ad-p53 gene therapy for head and neck squamous cell
carcinoma (HNSCC)—a cancer that accounts for about 10% of the 2.5
million annual new cancer patients in China.
The reasons for cancer to become a preferred area of application for gene
therapy are the significant unmet medical needs in cancer therapy, coupled
with the large size of its market. Additionally, the ethical acceptance of
gene therapy as a therapeutic solution also a contribution
12. CVS
Stem cell transplantation therapies that would accelerate
natural processes of postnatal collateral vessel formation, an
approach referred to as therapeutic angiogenesis
Coronary (CAD) and peripheral (PAD) artery disease
Direct intramyocardial injection of VEGF DNA using an
adenovirus vector in patients with otherwise inoperable
coronary artery disease and intractable angina pectoris
Rhumatoid arthritis- liposome, plasmid, RNAi
Alpha-1 antitrypsin (AAT) deficiency -is a hereditary disorder
associated with mutations in the SERPINA1 gene
13. Over 100 different alleles have been identified however the most common
disease-causing mutation
Others:-
14. RNA interference
RNA interference, also known as RNAi presents a new
approach to gene therapy by targeting specific genes and
down-regulating gene expression
One of the most potent forms of RNAi is small interfering
RNA, or siRNA
Small fragments of double stranded RNA, specific for a
particular gene target, are introduced to the cell
Specific hybridization between the naturally occurring
transcript and the induced siRNA (antisense portion)
instigates the destruction of the message.
directly on the transcriptional level of gene expression.
Therapeutically speaking, siRNA efficacy would be
determined by percent knock-down
BUT- this method is transient, requiring re-administration
15. Current scenario
The most promising gene-therapy concepts, at the
present time :
1. Direct killing of tumour cells with genes delivered by
Ad vectors for local management of cancer;
2. Delivery of naked DNA by injection or by the gene
gun for preventative vaccination against infectious
diseases;
3. Naked DNA delivery of genes promoting
angiogenesis for cardiovascular disorders; and
4. AV delivery for chronic disorders, such as
haemophilia and anaemia
16. Challenges
Short Lived- Hard to rapidly integrate therapeutic
DNA into genome and rapidly dividing nature of cells
prevent gene therapy for long time
Immune Response- leads to immune response
Increased response when a repeat offender enters
Viral Vectors- patient could have toxic, immune,
inflammatory response
also may cause disease once inside
Multigene Disorders- Heart disease, high blood
pressure, Alzheimer’s, arthritis diabetes,etc. need to
introduce more than one gene
May induce a tumor if integrated in a tumor
suppressor gene because of insertional mutagenesis
Ethical and moral challenges
Genes are carried on chromosomes and are the basic physical and functional units of heredity Genes are specific sequences of bases that encode instructions on how to make proteins When genes are altered so that the encoded proteins are unable to carry out their normal functions, genetic disorders result
characteristics: (1) a high capacity for self-renewal; (2) the potential for differentiation in multiple cell types; (3) the ability to be cultured ex vivo and used for tissue engineering; and (4) plasticity (transdifferentiation ability) (Vats et al., 2005). On the basis of their differentiating potential, stem cells can currently be classified into four categories: (1) totipotent, (2) pluripotent, (3) multipotent, and (4) oligopotent or monopotent. Totipotent stem cells have the potential to differentiate into cells of all three main germinal layers (the ectodermal, endodermal, and mesodermal) and embryo-derived tissues. Pluripotent stem cells have the ability to differentiate only into tissues derived from the ectoderm, endoderm and mesoderm. Multipotent stem cells can differentiate into tissue-specific progenitor cells within a given organ. For example, multipotent blood stem cell or hematopoietic stem cells can develop into red blood cells, white blood cells, or platelets. Oligopotent or monopotent stem cells can only give rise to one or few types of specialized cells. On the basis of their origin and biological properties, stem cells can also be classified as either (1) embryonic stem cells or (2) adult stem cells. Embryonic stem cells are derived from the inner layer mass of the blastocyst and can be harvested from three sources: aborted fetuses (cadaveric stem cells), embryos left over from in vitrofertilization (discarded embryos), and embryos created in the laboratory solely for the purpose of producing stem cells (research embryos). In vitro differentiation of human embryonic stem cells into cardiomyocytes has been demonstrated by Kehat et al. (Kehat et al., 2001). However, ethical issues have been raised against harvesting human embryonic stem cells, especially if this process requires destruction of an embryo. Other potential obstacles to using embryonic stem cells are that recipients often need to receive immunosuppressants, because embryonic stem cells are potentially allogenic and strongly immunogenic. Uncontrolled differentiation of embryonic stem cells may cause other problems, such as the development of cardiac or vascular neoplasm. Transplanted embryonic stem cells may form teratomas if some undifferentiated totipotent cells are still present. The formation of teratomas -i.e., tumors containing a mix of differentiated human cell types, including cells characteristic of the ectoderm, mesoderm, and endoderm-in severe combined immunodeficiency (SCID) mice after injection with human embryonic stem cells has been observed (Thomson et al., 1998). Adult stem cells are the undifferentiated cells that exist in a differentiated tissue or organ and that are capable of specializing into cells of the tissue or organ from which they originated. Their capacity for self-renewal allows tissues and organs to maintain functional stability. Sources of adult stem cells include not only regenerating tissues, such as the heart, adipose tissue, bone marrow, blood, liver;;’’’RNA interference (RNAi) is known to be a powerful means of sequence-specific gene silencing and would thus be a good candidate for this new strategy. RNAi was first reported in 1998 by Fire et al. who demonstrated that double-stranded RNA induced sequence-specific silencing of gene expression in nematode cells (
Hugocalled Human Gene Therapy Subcommittee, or HGTS n nih