NEW DRUGS AND

THERAPIES FOR
GENETIC DISEASES

SUBMITTED BY –
Farheen Khan
Roll no –19MBS007
MSc. Biosciences
1st year (2nd semester)
GENE THERAPY
There are two basic approaches to gene therapy: germline therapy and
somatic cell therapy.
Germ line gene therapy is where germ cells (sperm or egg) are
modified by the introduction of functional genes, which are integrated
into their genome. Therefore changes due to therapy would be heritable
and would be passed on to later generation.Germline therapy is usually
carried out by microinjection of a somatic cell followed by nuclear
transfer into an oocyte, and theoretically could be used to treat any
inherited disease.
Somatic cell therapy involves manipulation of cells, which either can
be removed from the organism, transfected, and then placed back in the
body, or transfected in situ without removal. The technique has most
promise for inherited blood diseases (e.g., hemophilia and
thalassaemia), with genes being introduced into stem cells from the
bone marrow, which give rise to all the specialized cell types in the
blood. The strategy is to prepare a bone extract containing several
billion cells, transfect these with a retrovirus-based vector, and then re-
implant the cells. Subsequent replication and differentiation of
transfectants leads to the added gene being present in all the mature
blood cells.Somatic cell therapy also has potential in the treatment of
lung diseases such as cystic fibrosis, as DNA cloned in adenovirus
vectors or contained in liposomes is taken up by the epithelial cells in
the lungs after introduction into the respiratory tract via an inhaler.
Gene therapy states and remains an experimental discipline and many
researches remain to be performed before the treatment will realize its
potential. Majority of the gene therapy trials are being conducted in
united States and europe, with only a modest number in other countries
including Australia. Scope of this approach is broad with potential in
treatment of diseases caused by single gene recessive disorders (like
cystic fibrosis, hemophilia, muscular dystrophy, sickle cell anemia
etc), acquired genetic diseases such as cancer and certain viral infections
like AIDS.
CANCER
The first FDA-approved gene therapy experiment in the United States
occurred in 1990 for a patient with severe combined immunodeficiency
disorder. Since then, many clinical trials have been conducted for
patients with cancer, using different approaches in gene therapy, with
successful results reported in patients with chronic lymphocytic
leukemia, acute lymphocytic leukemia, brain tumors, as well as others.
Several commercially approved medications for gene therapy were
released, including ONYX-15 (Onyx Pharmaceuticals) for refractory
head and neck cancer , human papilloma virus vaccine for the
prevention of cancer cervix (2006) and modified dendritic cells,
sipuleucel-T, for minimally symptomatic, castration-resistant metastatic
prostate cancer .
Most cancers result from activation of an oncogene that leads to tumor
formation, or inactivation of a gene that normally suppresses formation
of a tumor. In both cases a gene therapy could be envisaged to treat the
cancer. Inactivation of a tumor suppressor gene could be reversed by
introduction of the correct version of the gene by one of the methods
described above for inherited disease. Inactivation of an oncogene
would, however, require a more subtle approach, as the objective would
be to prevent expression of the oncogene, not to replace it with a non-
defective copy . One possible way of doing this would be to introduce
into a tumor a gene specifying an antisense version of the mRNA
transcribed from the oncogene. An alternative would be to introduce a
gene that selectively kills cancer cells or promotes their destruction by
drugs administered in a conventional fashion. This is called suicide
gene therapy and is looked on as an effective general approach to
cancer treatment, because it does not require a detailed understanding of
the genetic basis of the particular disease being treated. Many genes that
code for toxic proteins are known, and there are also examples of
enzymes that convert non-toxic precursors of drugs into the toxic form.
Introduction of the gene for one of these toxic proteins or enzymes into
a tumor should result in the death of the cancer cells, either immediately
or after drug administration .
Another approach is to use gene therapy to improve the natural killing
of cancer cells by the patient’s immune system, perhaps with a gene that
causes the tumor cells to synthesize strong antigens that are efficiently
recognized by the immune system. All of these approaches, and many
not based on gene therapy, are currently being tested in the fight against
cancer.In trials using a type of virus known as adenovirus, viral therapy
has shown encouraging results against several types of cancers,
including squamous cell cancers of the head and neck, and is being
tested as a preventive treatment for precancerous oral tissue. Trials
involving modified forms of herpes simplex virus have been conducted
in patients with malignant glioma (a form of brain cancer) and
colorectal cancer that has spread to the liver.
CAR T-cell therapy, which seeks to enhance the natural cancer-
fighting ability of patients’ own T cells, is one type of immunotherapy.
A sample of a patient’s T cells is collected and mixed with viruses
carrying several specific genes. The viruses deliver these genes to the T
cells’ nuclei, where they’re incorporated into the cells’ DNA. The genes
cause the T cells to express a special protein called a chimeric antigen
receptor, or CAR, on their surface. The CAR directs the T cell to the
tumor cell using a specific “address,” and the CAR T cell is then
equipped to rapidly destroy the cancer cell. When the cells, now called
CAR T cells, are infused into the patient, they seek out tumor cells and
then proliferate to generate many more cancer-killing cells.
Another form of immunotherapy involving gene therapy is cancer
vaccines. This approach involves collecting tumor cells from a patient
and engineering them with genes that cause them to be more
conspicuous to the immune system. The altered cells are then re-infused
into the patient along with an immune-stimulating compound. The
patient’s immune system launches a vigorous attack not only on the
newly-infused cancer cells but also on similar cells throughout the body.

SICKLE CELL ANEMIA

Sickle cell anemia is caused by a mutation in the HBB gene which
provides the instructions to make part of hemoglobin, the protein in red
blood cells that carries oxygen. Researchers are working on two
different strategies to treat sickle cell anemia with gene therapy. Both of
these strategies involve genetically altering the patient’s
own hematopoietic stem cells. These are cells in the bone marrow that
divide and specialize to produce different types of blood cells, including
the red blood cells.

One strategy is to remove some of the patient’s hematopoietic stem

cells, replace the mutated HBB gene in these cells with a healthy
copy of the gene, and then transplant those cells back into the
patient. The healthy copy of the gene is delivered to the cells using a
modified, harmless virus. These genetically corrected cells will then
hopefully repopulate the bone marrow and produce healthy, rather than
sickled, red blood cells.
The other strategy is to genetically alter another gene in the patient’s
hematopoietic stem cells so they boost production of fetal
hemoglobin — a form of hemoglobin produced by babies from about
seven months before birth to about six months after birth. This type of
hemoglobin represses sickling of cells in patients with sickle cell
anemia, but most people only produce a tiny amount of it after infancy.
Researchers aim to increase production of fetal hemoglobin in stem
cells by using a highly specific enzyme to cut the cell’s DNA in the
section containing one of the genes that suppress production of fetal
hemoglobin. When the cell repairs its DNA, the gene no longer works
and more fetal hemoglobin is produced.
Researchers at the National Institutes of Health have developed a new
and improved viral vector—a virus-based vehicle that delivers
therapeutic genes—for use in gene therapy for sickle cell disease. In
advanced lab tests using animal models, the new vector was up to 10
times more efficient at incorporating corrective genes into bone marrow
stem cells than the conventional vectors currently used, and has carrying
capacity of up to six times higher . The development of the vector could
make gene therapy for sickle cell disease much more effective and pave
the way for wider use of it as a curative approach for the painful, life-
threatening blood disorder .
The advent of genome engineering has expanded possible genetic
therapy options for many hematopoietic diseases like SCD . Several
other trials are under process to test their efficacy and safety of gene
therapy to replace the mutated HBB gene with a healthy HBB gene .
Researchers are investigating the use of Mozobil (plerixafor) in patients
with sickle cell anemia to increase the production of stem cells to be
used for gene therapy. This medication is already approved to treat
certain types of cancer. One trial is recruiting sickle cell anemia
patients in to donate bone marrow to be used in laboratory research to

develop gene therapy techniques. A study is examining the best way to

collect, process and store umbilical cord blood from babies with and
without sickle cell anemia. Cord blood contains abundant stem cells that
could be used in developing gene therapy for sickle cell anemia. Gene
addition trials has already shown that high-level expression of anti-
sickling β-globin can reverse the complications of the disease. With the
improvements in cell processing, viral vectors, and transduction
methods, we can anticipate validation of these encouraging results over
the short term, allowing broader application of potentially curative
strategies to patients with SCD .
The most elegant genetic medicine for a monogenic genetic disorder
would be to correct the mutation and thereby allow the production
of a normal protein under the endogenous regulatory signals of the
cell. An advance in this direction was the recent report of efficient
targeted gene correction using engineered zincfinger nucleases
(ZFNs). ZFNs target the interleukin 2 receptor-γc mutation that
causes X-linked severe combined immunodeficiency. In human cells
lines in culture, ZFNs corrected 18% of the primary blood cells and
5% of the T cells without any selection. The proposed strategy for
using ZFN strategies in the clinic would be for applications, such as
sickle cell anaemia, in which bone marrow stem cells could be
harvested from a patient, corrected ex vivo, and then returned to the
patient. Important challenges for this technology include how to
develop the ZFNs, managing the immune responses to a foreign
protein and achieving target specificity .

MUSCULAR DYSTROPHY
Muscular dystrophy refers to a group of more than 30 inherited diseases
that result in progressive weakening and wasting of muscles. These
diseases are caused by mutations in genes that are involved in the
production of proteins required for muscles to work properly.  
Currently, several kinds of gene therapies are being investigated to treat
muscular dystrophy, and possibly offer the chance of a cure. These
approaches are summarized below.
Exons are part of the gene that provides instructions to produce a
working protein. Exon skipping is the “patching” of that part of the
gene with missing or mutated exons, using short stretches of DNA
called antisense oligonucleotides (AO). This can lead to the production
of a truncated, albeit functional, protein to ease some of
the symptoms of muscular dystrophy. Exon skipping is currently
being evaluated in clinical trials for Duchenne muscular dystrophy
(DMD) and Becker muscular dystrophy (BMD) as these disorders are
caused due to deletion of exons at several places in the DMD gene.

Alternative strategy involves over-expression of compensating genes

either through gene transfer or by up regulating expression using
small molecules. Utrophin the paralog of dystrophin is highly
expressed in the fetal period. Over expression of utrophin, confers a
protective effect.. Encouraging result like improved muscle function
are obtained in mice with dystrophin deficiency when utrophin
transgenes are over expressed. Now researchers are trying to
discover new molecule which can upregulate the utrophin gene
expression upon administration.

Translarna (ataluren) is being studied in DMD and BMD patients to

reverse the effects of a so-called nonsense mutation that results in a
premature stop signal preventing the production of a full dystrophin
protein. Translarna is approved across most of Europe, Israel, and South
Korea to treat nonsense mutation DMD, but is not approved in the U.S.

In DMD Patients, Translarna Works by Bypassing ‘Stop’ Sign to Create Functional Protein

Gentamicin is an antibiotic used in the treatment of several bacterial

infections and investigated as a stop codon readthrough therapy for
DMD. A study found that gentamicin was effective in raising
dystrophin levels and lowering serum creatine kinase levels.
Adeno-associated viruses (AAVs) are a class of modified viruses that
are commonly used as vectors, or transport agents, to deliver
functioning genes directly into specific tissues. Three such therapies that
use AAVs are currently being evaluated. 
GALGT2 introduces the GALGT2 gene in skeletal and heart muscles to
increase the production of other proteins required for muscle cell
function.
SRP-9001 micro-dystrophin introduces a gene coding for
microdystrophin — a protein that is functionally similar to dystrophin
but smaller in size — into the heart and skeletal muscles.
SGT-001 also aims to introduce a gene coding for microdystrophin into
muscle cells. OurOur cells have evolved mechanisms to prevent
intrusion of foreign molecules. To overcome this we use vectors, or
carriers, to carry the new gene into the cell. Currently, the most
promising approach is based on the use of a harmless virus
called Adeno-associated virus (AAV) as a vector. Viruses have
evolved to recognise certain cells and then insert themselves via the cell
membrane, and deliver their genetic material into the cell (usually
making us feel unwell). However, if scientists remove the unwanted,
disease-causing genes and replace them with appropriate beneficial
genes, they could restore gene expression. This is the basis of the ‘gene
therapy’ that is causing great excitement at the moment.
RNAi is the process of inhibiting, or blocking, the function of the
messenger RNA (mRNA) produced in a gene. mRNA contains
instructions from the gene ultimately required to make a protein. BB-
301 is an experimental RNAi therapy that is being investigated to
treat oculopharyngeal muscular dystrophy (OPMD).
BB-301 uses RNAi to prevent the production of an abnormal PABPN1
protein via mRNA from the mutated PABPN1 gene. At the same time, it
introduces a healthy copy of the PABPN1 gene into a cell.

SEVERE COMBINED IMMUNODEFICIENCIES

Severe combined immunodeficiency (SCID, pronounced “skid”) is a
rare, fatal syndrome of diverse genetic cause in which there is combined
absence of Tlymphocyte and B-lymphocyte function (and in many
cases also natural killer, or NK lymphocyte function). These defects
lead to extreme susceptibility to serious infections. There are currently
twelve known genetic causes of SCID. Although they vary with respect
to the specifi c defect that causes the immunodefi ciency, some of their
laboratory fi ndings and their pattern of inheritance, these all have
severe defi ciencies in both T cell and B cell function.
Severe Combined Immunodeficiency Disease (SCID) is also due to a
defective gene for Adenosine Deaminase (ADA). A retrovirus, which
is capable of transferring it's DNA into normal eukaryotic cells
(transfection), is engineered to contain the normal human ADA gene.

Isolated T-cell stem line cells from the patient are exposed to the

retrovirus in cell culture, and take up the ADA gene. Reimplantation of
the transgenic cells into the patient's bone marrow establishes a line of
cells with functional ADA, which effecitvely treats SCID

SPECIFIC THERAPY
Immunoglobulin (IVIG) replacement therapy should be given to SCID
infants who are more than 3 months of age and/or who have already had
infections. Although immunoglobulin therapy will not restore the
function of the defi cient T-cells, it does replace the missing antibodies
resulting from the B-cell defect and is, therefore, of some benefi t. For
patients with SCID due to ADA deficiency, replacement therapy with a
modifi ed form of the enzyme (from a cow, called PEG-ADA) has been
used with some success. The immune reconstitution effected by PEG-
ADA is not a permanent cure and requires 2 subcutaneous injections
weekly for the rest of the child’s life. PEG-ADA treatment is not
recommended if the patient has an HLA-matched sibling available as a
donor for a marrow transplant. The most successful therapy for SCID is
immune reconstitution by bone marrow transplantation. Bone marrow
transplantation for SCID is best performed at medical centers that have
had experience with SCID and its optimal treatment and where there are
pediatric immunologists overseeing the transplant. In a bone marrow
transplant, bone marrow cells from a normal donor are given to the
immunodeficient patient to replace the defective lymphocytes of the
patient’s immune system with the normal cells of the donor’s immune
system. The goal of transplantation in SCID is to correct the immune
dysfunction. This contrasts with transplantation in cancer patients,
where the goal is to eradicate the cancer cells and drugs suppressing the
immune system are used heavily in that type of transplant.
The ideal donor for a SCID infant is a perfectly HLA-type matched
normal brother or sister. Lacking that, techniques have been developed
over the past three decades that permit good success with half-matched
related donors (such as a mother or a father). Pre-transplant
chemotherapy is usually not necessary. Several hundred marrow
transplants have been performed in SCID infants over the past 30 years,
with an overall survival rate of 6070%. However, the outcomes are
better if the donor is a matched sibling (>85% success rate)and if the
transplant can be performed soon after birth or less than 3.5 months of
life (>96% survival even if only half-matched). HLA matched bone
marrow or cord blood transplantation from unrelated donors has also be
used successfully to treat SCID.
Finally, another type of treatment that has been explored over the past
two decades is gene therapy. There have been successful cases of gene
therapy in both X-linked and ADA-deficient SCID. However, research
in this area is still being conducted to make this treatment safer .
Gene therapy for the X-linked form of SCID (XSCID) was attempted in
two infants. XSCID is due to genetic defects of an X-linked gene
encoding a component of the cell surface receptor for five different
growth factors involved in lymphocyte development and activation, the
common cytokine receptor gamma chain or gc.
Bone marrow was collected from the infants and exposed to a retroviral
vector carrying a normal copy of the human gc gene. The marrow cells
were returned to the infants by intravenous infusion. Over the
subsequent months, both infants developed normal numbers of T
lymphocytes and natural killer (NK) cells, which showed evidence of
immunologic function. Both infants have remained in good health, free
of opportunistic infections, growing and developing without protective
isolation. Initial evidence suggests they have also developed B
lymphocyte function with the presence of protective levels of
antibodies, although the number of B lymphocytes remains low. The
tempo at which T lymphocyte numbers and function developed is
essentially identical to the response to transplantation of genetically
normal hematopoietic stem cells from the bone marrow of a healthy,
HLA-matched sibling donor. There was near-normalization of T cell
numbers within 2 to 3 months, which reflects the minimal time it takes
for a human HSC or lymphoid progenitor to enter the thymus, undergo
maturation and enter the periphery, based upon observation of SCID
patients who receive T cell depleted bone marrow transplant.

REFERENCES
 GENE CLONING AND DNA ANALYSIS An
Introduction T.A. BROWN Sixth Edition
 https://en.m.wikipedia.org/wiki/Gene_therapy

 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC157048

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 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC476008
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