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Nucleic Acid-Based Therapeutics

Prominent category of biomolecule in living

cells

Related to DNA and RNA

DNA
the genetic information in living cells

RNA
the genetic information in some
viruses

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Is polynucleotides

A nucleotide consis of:
- phosphate group,
- pentose (five-carbon sugar)
ribose
& deoxyribose
- nitrogenous base
purine (A & G),
pirimidine (T ,C, U)

(b) Adenosine 5-monophosphate

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Throughout the 1980s and early 1990s

biopharmaceutical refer to proteins or nucleic acid
based therapeutic.

Antisense technology (including RNAi) and aptamer

technology
potential to revolutionize medical practice
2007
3 nucleic acid products were approved

antisense-based product (Vitravene),
aptamer (Macugen), gene therapy product
(Gendicine, approved only in China).

165 protein products approved

Cell-based medicines - a small number
were approved

 Antiviral drug
to treat cytomegalovirus retinitis

(CMV) in immunocompromised patients (patients
with AIDS).

 The first antisense antiviral approved by the FDA for

CMV in Aug 1998.

 A synthetic 21 member oligonucleotide:

5'-GCG TTT GCT CTT CTT CTT GCG-3

 Link to phosphorothioate
resistant to degradation

by nucleases

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Mechanism
blocks translation of viral mRNA by binding
to the complementary sequence of the
mRNA transcribed from the coding segment
of CMV gene.

Administration
Intraocular injection
(6.6 mg/mL)

Anti-angiogenic
to treat neovascular age-related

macular degeneration (AMD).

Discovered by Gilead Sciences (USA). Marketed by

Pfizer.

Approval by FDA
December 2004.

PEGylated anti-VEGF aptamer - single strand nucleic

acid binds specifically to a particular target (VEGF165)

Administration intravitreal injection (0.3 mg/6weeks)

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Recombinant adenovirus (rAd-p53)
express

wildtype-p53.

To treat tumor patients with p53 genes mutation
in head and neck squamous cell carcinoma and
various other tumors.

THE FIRST gene therapy product approved for
clinical use in humans.

Manufactured by Shenzhen SiBiono GeneTech

Types:

Antisense technology

Oligonucleotides

Aptamers

Application is widely known as:

Gene therapy

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So, what about the Gene Therapy

implementation ??

Theoretically straightforward, but difficult

to achieve in practice

The principles:

introduction of a gene into the genetic
complement of a cell

the expressed gene wish to achieve a
therapeutic goal

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Potential as a curative approach for :

inborn errors of metabolism

conditions caused by defective copy of
specific gene (s)

Other roles:
to study the molecular basis of various diseases
(cancer, AIDS, and neurological conditions) and
how to combat these diseases.

2/3 of gene therapy trials
dedicated to treat

cancer

The first trial
USA in 1989.

Later, 1200 clinical studies were undertaken

worldwide 67 % undertook in the USA, the
remaining in Europe (UK and in Germany).

Limited studies have revealed a therapeutic

benefit to the patient.

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Cancer (various forms)

Cystic fibrosis
Familial hypercholesterolemia
AIDS
Haemophilia
Severe Combined Immunodeficiency Diseases (SCID)
Gauchers Disease
Purine nucleoside phosphorylase deficiency
Rheumatoid Arthritis (RA)
Peripheral vascular Disease
Alpha-1 antitrypsin deficiency

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US patient died in 1999 as a result of

participating in one such trial.

The ensuing FDA investigation
6 deaths

caused by clinical trial treatments - were
unreported, only a fraction of serious adverse
effects had been reported.

As a result, regulation and monitoring of gene

therapy trials has been increased.

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(a) Therapeutic nucleic acid ENTRY to the cell

cytoplasm
vector help.
(b) Nucleic acid transfer to the nucleus of
recipient cell.
(c) Expression of foreign gene (whether
integrated or not)
result the desired
protein product.
Regulatory elements of the nucleic acid
transferred may be designed to ensure the
protein product is retained within the cell, or
is exported from the cell, as is necessary.

The desired gene
packaged into a vector

system to deliver the gene inside the
recipient cells.

Vectors of gene transfer viral and non-viral.

Once assimilated by the cell, the

exogenous nucleic acid must be delivered
to the nucleus.

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(a) Invitro gene therapy

(b) In situ gene therapy

vector injection
nearby the target cells
(a) In vivo gene therapy

vector IV injection

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Removal of the target cells from the body.

The cells cultured in vitro and incubated with vector
containing the nucleic acid to be delivered.

The genetically altered cells reintroduced into the
patients body.

To be success
the target cells must be easy to

remove from the body, and reintroduce into the body.

Such in vitro approaches have successfully been

undertaken utilizing various body cell types, including
blood cells, stem cells, epithelial cells, muscle cells
and hepatocytes.

Direct administration of the nucleic-acid-

containing vector to the target cell in the
body.

Examples:

direct injection of vectors into a tumour
mass

aerosol injection of vectors (e.g. containing
the cystic fibrosis gene) to respiratory tract
epithelial cells.

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Is not always feasible.

Feasible if : the target cells is localized at one specific area
What about blood cells ? Is it feasible to perform in situ gene
therapy? Then.?

Intensive efforts

the inclusion of antibody on vector surface, which specifi
cally binds a surface-antigen uniquely associated with the
target cell, would allow selective delivery.

manipulate vector to display a specific hormone that
would bind only to the cells that display the hormone
receptor.

Retroviral vectors

24% of all gene therapy clinical trials

retroviral vectors as gene delivery systems.

Retroviruses : enveloped viruses.

Consists of ssRNA - 58 kb.
Upon entry into sensitive cells, the viral RNA is
reverse transcribed and yields dsDNA.
dsDNA subsequently integrates into the host
cell

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Vehicle Advantages Disadvantages

NONVIRAL

Naked DNA Ease of production Low efficiency

 No DNA size limitation

Liposome-
Liposome-  Ease of production Low efficiency
DNA
 No DNA size limitation
 Low immune reaction

VIRAL
Retrovirus  Ease of production Transfer to dividing cells only

 Efficient DNA transfer Random DNA integration

 Stable expression DNA transfer size limited
 Low immune reaction

Adenovirus  Ease of production Host immune reaction

 Efficient DNA transfer Transient expression
 Transfer to non dividing cells DNA transfer size limited

Gene Therapy: Vectors

Plasmid Based Vectors Virus-

Virus-Based Vectors
Plasmid Retrovirus
Plasmid with liposome Adenovirus
Plasmid linked to ligand Adeno--associated virus
Adeno
Herpes simplex virus

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Inherited genetic defect

E.g. introduction of the cystic fibrosis trans membrane
conductance regulator (CFTR) gene (the cystic fibrosis gene)
into the airway epithelial cells of cystic fibrosis sufferers

Novel function of the recipient cell

E.g. introduction of a novel gene into white blood cells
whose protein product is capable of in some way interfering
with HIV replication. Such an approach might prove an
effective therapeutic strategy for the treatment of AIDS

Gene product duration of expression

- Long-term expression of the inserted gene would be
required.
- Short term expression should be sufficient (e.g. some
forms of cancer therapy or the use of gene therapy to
deliver a DNA-based vaccine)

Regulation of expression
Choice of target cells
e.g. treatment of the genetic condition, familial
hypercholesterolaemia
insertion of the gene coding for
the low-density lipoprotein receptor specifically in
hepatocytes.

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Short-lived nature of gene therapy

Before gene therapy can become a permanent cure:
- must remain functional
- the cells containing the therapeutic DNA must be long-lived and
stable.

Problems: the cells rapidly dividing naturally in many cells

Prevent gene therapy from achieving any long-term benefits

Hence, patients will have to undergo multiple rounds of gene
therapy.
Immune response

when a foreign object is introduced into human tissues
the immune
system will attack the invader
it makes gene theraphy difficult to be given
repeatedly to the patient.

Current gene therapy is experimental and has not proven very

successful in clinical trials.
Little progress has been made since the first gene therapy clinical trial
began in 1990.
In 1999, gene therapy suffered from a death of 18-year-old Jesse
Gelsinger. He was participating in a gene therapy trial for ORNITHINE
TRANSCARBOXYLASE DEFICIENCY (OTCD). He died from multiple
organ failures four days after starting the treatment. His death is
believed to have been triggered by a severe immune response to the
adenovirus carrier.

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THANK YOU

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