Assigment No:-01

Genetic Engineering in Pharmaceutical Product Development.

Recombinate Insulin:

The hormone insulin is produced by the (J-cells of islets of Langerhans of pancreas.

Human insulin contains 51 amino acids, arranged in two polypeptide chains. The chain
A has 21 amino acids while B has 30 amino acids. Both are held together by disulfide
bonds.

Diabetes mellitus:

Diabetes mellitus affects about 2-3% of the general population. It is a genetically linked
disease characterized by increased blood glucose concentration (hyperglycemia). The
occurrence of diabetes is due to insufficient or inefficient (incompetent) insulin. Insulin
facilitates the cellular uptake and utilization of glucose for the release of energy.

In the absence of insulin, glucose accumulates in the blood stream at higher

concentration, usually when the blood glucose concentration exceeds about 180 mg/dl,
glucose is excreted into urine. The patients of diabetes are weak and tired since the
production of energy (i.e. ATP) is very much depressed.

The more serious complications of uncontrolled diabetes include kidney damage

(nephropathy), eye damage (retinopathy), nerve diseases (neuropathy) and circulatory
diseases (atherosclerosis, stroke). In fact, diabetes is the third leading cause of death
(after heart disease and cancer) in many developed countries.

In the early years, insulin isolated and purified from the pancreases of pigs and cows
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was used for the treatment of diabetics. There is a slight difference (by one to three
amino acids) in the structure of animal insulin compared to human insulin. This
resulted in allergy in some of the diabetics when animal insulin was administered.

Another problem with animal insulin is that large number of animals have to be
sacrificed for extracting insulin from their pancreases. For instance, about 70 pigs
(giving about 5 kg pancreatic tissue) have to be killed to get insulin for treating a single
diabetic patient just for one year!
Production of recombinant insulin:

Attempts to produce insulin by recombinant DNA technology started in late 1970s. The
basic technique consisted of inserting human insulin gene and the promoter gene of lac
operon on to the plasmids of E. coli. By this method human insulin was produced. It was
in July 1980, seventeen human volunteers were, for the first time, administered
recombinant insulin for treatment of diabetes at Guy’s Hospital, London.

And in fact, insulin was the first ever pharmaceutical product of recombinant DNA
technology administered to humans. Recombinant insulin worked well, and this gave
hope to scientists that DNA technology could be successfully employed to produce
substances of medical and commercial importance. An approval, by the concerned
authorities, for using recombinant insulin for the treatment of diabetes mellitus was
given in 1982. And in 1986, Eli Lilly Company received approval to market human
insulin under the trade name Humulin.

Technique for recombinant insulin production:

The original technique of insulin synthesis in E. coli has undergone several changes, for
improving the yield, e.g. addition of signal peptide, synthesis of A and B chains
separately etc. The procedure employed for the synthesis of two insulin chains A and B
is illustrated in Fig. 15.1. The genes for insulin A chain and B chain are separately
inserted to the plasmids of two different E. coli cultures.

The lac operon system (consisting of inducer gene, promoter gene, operator gene and
structural gene Z for β-galactosidase) is used for expression of both the genes. The
presence of lactose in the culture medium induces the synthesis of insulin A and B
chains in separate cultures. The so formed insulin chains can be isolated, purified and
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joined together to give a full-pledged human insulin.

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Second generation recombinant insulin’s:

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After injecting the insulin, the plasma concentration of insulin rises slowly. And for this
reason, insulin injection has to be done at last 15 minutes before a meal. Further,
decrease in the insulin level is also slow, exposing the patients to a danger of hyper-
insulinemia. All this is due to the existence of therapeutic insulin as a hexamer (six
molecules associated), which dissociates slowly to the biologically active dimer or
monomer.
Attempts have been made in recent years to produce second generation insulin by site-
directed mutagenesis and protein engineering. The second generation recombinant
proteins are termed as muteins. A large number of insulin muteins have been
constructed with an objective of faster dissociation of hexamers to biologically active
forms. Among these is insulin lispro, with modified amino acid residues of the B-chain
of insulin. Insulin lispro can be injected immediately before a meal as it attains the
pharmacologically efficient levels very fast.

Chemically altered porcin insulin:

As already stated, porcin (pig) insulin differs from human insulin just by one amino
acid-alanine in place of threonine at the C-terminal and of B-chain of human insulin.
Biotechnologists have developed methods to alter the chemical structure of porcin
insulin to make it identical to human insulin. And this chemically modified porcin
insulin can also be employed for the treatment diabetes mellitus.

Human Growth Hormone:

Growth hormone is produced by the pituitary gland. It regulates the growth and
development. Growth hormone stimulates overall body growth by increasing the cellular
uptake of amino acids, and protein synthesis, and promoting the use of fat as body fuel.

Insufficient human growth hormone (hGH) in young children results in retarded

growth, clinically referred to as pituitary dwarfism. The child usually is less than four
feet in height, and has chubby face and abundant fat around the waist.

Traditional treatment for dwarfism:

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The children of pituitary dwarfism were treated with regular injections of growth
hormone extracted from the brains of deceased humans. It may be noted that only
human growth hormone is effective for treatment of dwarfism. (This is in contrast to
diabetes where animal insulin’s are employed).

At least eight pituitary glands from cadavers must be extracted to get hGH adequate for
treating a dwarf child just for one year! And such treatment has to be continued for 8-10
years!! Further, administration hGH isolated from human brains exposes the children to
a great risk of transmitting the cadaver brain diseases (through virus or viral-like
agents) e.g. Creuzfeldt- Jacob (CJ) syndrome characterized by convulsions, wasting of
muscle etc.

Production of recombinant hGH:

Biotechnologists can now produce hGH by genetic engineering. The technique adopted
is quite comparable with that of insulin production. The procedure essentially consists
of inserting hGH gene into E. coli plasmid, culturing the cells and isolation of the hGH
from the extracellular medium.

Limitation in hGH production:
The hGH is a protein comprised of 191 amino acids. During the course of its natural
synthesis in the body, hGH is tagged with a single peptide (with 26 amino acids). The
signal peptide is removed during secretion to release the active hGH for biological
functions. The entire process of hGH synthesis goes on in an orderly fashion in the body.

However, signal peptide interrupts hGH production by recombinant technology. The

complementary DNA (cDNA) synthesized from the mRNA encoding hGH is inserted
into the plasmid. The plasmid containing E. coli when cultured, produces full length
hGH along with signal peptide. But E. coli cannot remove the signal peptide.

Further, it is also quite difficult to get rid of signal peptide by various other means.
Theoretically, cDNA encoding signal peptide can be cut to solve these problems.
Unfortunately, there is no restriction endonuclease to do this job, hence this is not
possible.

A novel approach for hGH production:

Biotechnologists have resolved the problem of signal peptide interruption by a novel
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approach (Fig. 15.2). The base sequence in cDNA encoding signal peptide (26 amino
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acids) plus the neighbouring 24 amino acids (i.e a. total 50 amino acids) is cut by
restriction endonuclease ECoRI.
Now a gene (cDNA) for 24 amino acid sequence of hGH (that has been deleted) is
freshly synthesized and ligated to the remaining hGH cDNA. The so constituted cDNA,
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attached to a vector, is inserted into a bacterium such as E. coli for culture and
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production of hGH. In this manner, the biologically functional hGH can be produced by
DNA technology. Recombinant hGH was approved for human use in 1985. It is
marketed as Protropin by Gene-tech Company and Humatrope by Eri Lilly Company.

Controversy over the use of hGH:

Recombinant hGH can be administered to children of very short stature. It has to be
given daily for many years with an annual cost of about $ 20,000. Some workers have
reported substantial increase in the height of growth retarded children.
One group of workers observed that the normal growth pattern in children was not
restored on hGH administration, although there was an initial spurt. Another big
question raised by the opponents of hGH therapy is whether it is necessary to consider
short stature as a disorder at all for treatment!

Use of recombinant growth hormone for farm animals:

Recombinant GH is now available for administration to farm animals to promote early
growth and development. Such farm animals yield linear meat, besides increased milk
production. However, use of GH in farm animals is a controversial issue.

Clotting Factor VIII:

The clotting factor VIII is required for proper blood clotting process. A genetic defect in
the synthesis of factor VIII results in the disorder hemophilia A. This is a sex-linked
disease (incidence 1 in 10,000 males) transmitted by females affecting males. The
victims have prolonged clotting time and suffer from internal bleeding.

Traditional treatment for hemophilia A:

Clotting factor VIII was isolated from the whole blood and administered to the patients
of hemophilia A. This approach requires large quantities of blood. Another problem is
the risk of transmission of certain diseases like AIDS to the hemophiliacs.

Production of recombinant factor VIII:

The gene for the formation of factor VIII is located on X chromosome. It is a complex
gene of 186 kb (i.e., 186,000 base pairs) in size, organized into 26 exons of varying
length. In between the exons, many introns are present. The introns vary in their size,
starting from 200 base pairs to as high as 32,000 base pairs.

Biotechnologists were able to isolate mature mRNA (containing only exons and no
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introns) that is responsible for the synthesis of factor VIII. This mRNA contains 9,000
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bases and synthesizes the protein, factor VIII. Factor VIII contains 2332 amino acids,
with carbohydrate molecules attached at least at 25 sites.

DNA technologists synthesized the complementary DNA (cDNA) for mature mRNA of
factor VIII. This cDNA can be inserted into mammalian cells or hamster kidney cells for
the production of recombinant factor VIII. Since 1992, factor VIII is available in the
market. It is produced by Genetics Institute in Cambridge, Massachusetts and sold as
Recombinant while Miles Laboratories sell under the trade name Kogenate.
Type # 2. Therapeutic Agents for Human Diseases:
Biotechnology is very useful for the production of several therapeutic products for
treating human diseases. A selected list of rDNA-derived therapeutic agents along with
trade names and their uses in humans are given in Table 15.3.

Some of these are described above (under human protein replacements)

while the remaining are discussed below:

Gene Therapy:

A gene is a long segment of the molecule deoxyribonucleic acid (DNA). This segment,
composed of minute subunits called nucleotide bases, serves as the blueprint for
manufacturing a single protein or enzyme needed for the structure or function of cells
(Griffiths et al., 2000). In humans, genes are compressed and bundled into a set of 23
pairs of chromosomes which stabilize and protect the DNA. Any tiny error in the
arrangement of a genes nucleotide bases can lead to the production of a protein or
enzyme that functions improperly, disrupting cellular biological activities such as codons
for proteins or enzymes production (Isenbarger et al., 2008). Genes correspond to regions
within DNA which is composed of a chain of four different types of nucleotides: adenine,
guanine, thymine and cytosine. The sequence of these nucleotides is the genetic
information organisms inherit DNA naturally occurs in a double stranded from, with the
nucleotide base pairs on each strand complementary to each other. Each strand can act as
a template for creating a new partner strand. This is the physical method for making
copies of genes that can be inherited. Viruses on the other hand use a similar molecule
ribonucleic acid, RNA, instead of DNA as their genetic material (Hershey and Chase,
1952). The nucleotides sequence in a gene is translated by cells to produce a chain of
amino acids; the order of amino acids in a protein corresponds to the order of nucleotides
in the gene. The relationship between nucleotide sequence and amino acids is known as
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the genetic code. The types of amino acids in a protein determine how it folds into a
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threedimensional shape. This structure is in turn responsible for the protein’s function. A
change to the DNA in a gene can change a protein’s amino acid changing its shape and
function; this can have a dramatic effect on the whole organism (Gura, 1999).

Gene Expression:

Genes generally express their functional effect through the production of proteins which
are complex molecules responsible for most functions in the cells. Proteins are chains of
amino acids, and the DNA sequence of a gene (through an RNA intermediate) is used to
produce a specific protein sequence (Mayeux, 2005). The process called transcription
begins with the production of an RNA molecule with a sequence that matches the gene’s
DNA sequence. The messenger RNA (mRNA) molecule is then used to produce a
corresponding amino acid sequence through a process called translations. Each group of
three nucleotides in the sequence called a codon corresponds either to one of the twenty
possible amino acids in a protein or an

Gene Regulation:

The genome of a given organism contains thousands of genes, but not all these genes
need to be active at any given moment. A gene is expressed when it is being transcribed
into mRNA (and translated into protein), and there exist many cellular methods of
controlling the expression of genes such that proteins are produced only when needed by
the cell. Transcription factors are regulatory proteins that bind to the start of genes, either
promoting or inhibiting the transcription of the gene (Wolf et al., 2002). Within the
genome of Escherichia coli bacteria, for example, there exists a series of genes necessary
for the synthesis of the amino acid tryptophan. Once tryptophan is already available in
the cell, the genes for tryptophan synthesis are no longer needed. The presence of
tryptophan directly affects the activity of the genes. Tryptophan molecules bind to the
tryptophan repressor (a transcription factor), changing the repressor's structure such that
the repressor binds to the genes. The tryptophan repressor blocks the transcription and
expression of the genes, thereby creating negative feedback regulation of the tryptophan
synthesis process (Yang, 2007). Differences in gene expression are especially clear
within multicellular organisms where cells all contain the same genome but have very
different structures and behaviors due to the expression of different sets of genes. All the
cells in a multicellular organism derive from a single cell, differentiating into variant cell
types in response to external and intercellular signals and gradually establishing different
patterns of gene expression to create different behaviors. As no single gene is responsible
for the development of structures within multicellular organisms, these patterns arise
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from the complex interactions between many cells (Yoon et al., 1996). Within eukaryotes
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there exist structural features of chromatin that influence the transcription of genes, often
in the form of modifications to DNA and chromatin that are stably inherited by daughter
cells. These features are called "epigenetic" because they exist "on top" of the DNA
sequence and retain inheritance from one cell generation to the next.
MATERIALS AND METHODS

A comprehensive internet search of literature on gene therapy was undertaken using

Google search. Literatures recovered were analyzed in pros and relevant cited tables and
figures adopted. RESULT AND DISCUSSION Types of Gene Therapy

1. Germline Gene Therapy:

Germline gene therapy involves the modification of germ cells (gametes) that will pass
the change on to the next generation. With germline therapy, genes sequence can be
corrected in the egg or the in sperm that is being used to conceive. The child that results
would be spared certain genetic problems that might otherwise have occur(Cole-Strauss
every cell descends from the fertilized egg, every cell in the offspring transplanted gene.
This would be a far more effective way of transferring genes than the ones presently used
in somatic cell therapies, where genes into the cells of children or adults usually enter
only a small portion of the person’s cells (Cheng et al., based on the effective delivery of
the corrective genes and to do this, scientists have developed gene delivery vehicles
called vectors. These vectors encapsulate ther delivery into the target vectors currently in
use are based on attenuated or modified versions of viruses. Plasmids, which are circular
pieces of DNA extracted from (restricti "digest" DNA at designated nucleotide locations
along the DNA chain). There are different types of restriction enzymes, each being
specific to the location of the DNA chain that it will cut. Gene therapy, physiological
applications, problems a Strauss et al., every cell descends from the fertilized egg, every
cell in the offspring transplanted gene. This would be a far more effective way of
transferring genes than the ones presently used in somatic cell therapies, where genes into
the cells of children or adults usually enter only a small portion of the person’s cells and
eventually stop functioning et al., 1994). The technology of gene therapy is based on the
effective delivery of the corrective genes and to do this, scientists have developed gene
delivery vehicles called vectors. These vectors encapsulate ther applications, problems a
Animal Research International (201 et al., 1999). In living organisms, every cell descends
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from the fertilized egg, every cell in the offspring transplanted gene. This would be a far
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more effective way of transferring genes than the ones presently used in somatic cell
therapies, where genes into the cells of children or adults usually enter only a small
portion of the and eventually stop functioning 1994). The technology of gene therapy is
based on the effective delivery of the corrective genes and to do this, scientists have
developed gene delivery vehicles called vectors. These vectors encapsulate therapeutic
genes for delivery into the targeted cells. Many of the vectors currently in use are based
on attenuated or modified versions of viruses. Plasmids, which are circular pieces of
DNA extracted from
2. Somatic Gene Therapy: Somatic gene therapy is the transfer of genes into the
somatic cells of the patient, such as cells of the bone marrow, and hence the new DNA
does not enter the eggs or sperm. The genes transferred are usually normal alleles that
could ‘correct’ the mutant or disease alleles of the recipient. The technique of somatic
gene therapy involves inserting a normal gene into the appropriate cells of an individual
affected with a genetic disease, thereby permanently correcting the disorder (Neuman et
al., 1982). The targeted cells may be bone marrow cells, which are easily isolated and re-
implanted. Bone marrow cells continue to divide for a person's whole life to produce
blood cells, so this approach is useful only if the gene you want to deliver has a biological
role in the blood. Delivery of a gene that has a biological role in the lungs, muscle, or
liver would have to occur within those targeted organs. In many cases,

Variations of the PCR Technique in Gene Therapy

Allele-specific PCR:

a diagnostic or cloning technique based on single-nucleotide polymorphisms (SNP)

(single-base differences in DNA). SNP requires prior knowledge of a DNA sequence,
including differences between alleles, and uses primers whose 3' ends encompass the
SNP. PCR amplification under stringent conditions is much less efficient in the presence
of a mismatch between template and primer, so successful amplification with an SNP-
specific primer signals presence of the specific SNP in a sequence (Lawyer et al., 1993).

Asymmetric PCR: preferentially amplifies one DNA strand in a double-stranded DNA

template. It is used in sequencing and hybridization probing where amplification of only
one of the two complementary strands is required. PCR is carried out normally, but with
excess of the primer for the strand targeted for amplification. Because of the slow
(arithmetic) amplification later in the reaction after the limiting primer has been used up,
extra cycles of PCR are required. A recent modification of this process, called Linear-
After-The-Exponential-PCR (LATE-PCR) uses a limiting primer with a higher melting
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temperature (Tm) than the excess primer to maintain reaction efficiency as the limiting
primer concentration decreases mid-reaction (Pierce and Wangh, 2007).
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Multiplex PCR: is the use of multiple primer sets within a single PCR mixture to produce
amplicons of varying sizes that are specific to different DNA sequences. By targeting
multiple genes at once, additional information may be gained from a single test-run that
otherwise would require several times the reagents and more time to perform. Annealing
temperatures for each of the primer sets must be fully optimized to work correctly within
a single reaction and amplicon sizes. That is, their base pair length should be different
enough to form distinct bands when visualized by gel electrophoresis (Pierce and Wangh,
2007; Isenbarger et al., 2008).

Nested PCR: increases the specificity of DNA amplification, by reducing background

arising from non-specific amplification of DNA. Two sets of primers are used in two
successive PCRs. In the first reaction, one pair of primers is used to generate DNA
products, which besides the intended target, may still consist of nonspecifically amplified
DNA fragments. The product(s) are then use in a second PCR with a set of primers
whose binding sites are completely or partially different from and located 3' of each of
the primers used in the first reaction. Nested PCR is often more successful in specifically
amplifying long DNA fragments than conventional PCR, but it requires more detailed
knowledge of the target sequences (Sarker et al., 1990; Pierce and Wangh, 2007).

Nanoparticles Transgenic technology has been widely used in breeding new plant and
animal varieties. Effective gene delivery into the target cells is an essential step in these
practices (Jaroslav et al., 2002). Both viral and nonviral vectors have been used for gene
delivery especially with regards to gene therapy. Nanoparticles as gene vectors, due to
their reduced immunogenicity, improved safety and the ability to carry larger DNA loads
has become an attractive alternative in gene delivery. Cationic liposomes have been
widely used as non-viral gene vectors, but, because of its cell-specific, easily inactivation
of serum protein, and other reasons, its scope of application is also limited (Mehrdad et
al.,

Applications of Gene Therapy

Genetic engineering is an natural scientists. Genes and other genetic information from a
wide range of organisms are trans modification, creating genetically modified bacteria in
the process. Bacteria are cheap, easy to grow, multiply quickly, relatively easy to
transform and can be stored at indefinitely. Once a gene is isolat stored inside the bacteria
providing an unlimited supply for research (Lodish medicine, agriculture and in the
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production of genetically engineered organisms in order to discover the functions 2005).

This could be the effect on the phenotype of the organism, where the gene is expressed or
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what other genes it interacts with.

1. Physiological application of gene therapy is in treating type I diabetes. Current

research used an adenovirus as a vector to deliver the gene for hepatocyte growth factor
(HGF) to pancreatic islet cells removed from rats. process. Bacteria are cheap, easy to
grow, multiply quickly, relatively easy to transform and can be stored at indefinitely.
Once a gene is isolat stored inside the bacteria providing an unlimited supply for research
(Lodish Gene therapy finds applications in medicine, agriculture and in the production of
genetically engineered organisms in order to discover the functions of certain genes
(Mayeux, 2005). This could be the effect on the phenotype of the organism, where the
gene is expressed or what other genes it interacts with. Physiological applications
application of gene therapy is in treating type I diabetes. Current research used an
adenovirus as a vector to deliver the gene for hepatocyte growth factor (HGF) to
pancreatic islet cells removed from rats. They injected the altered cells into diabetic rats
and, within a day, the rats were controlling their blood glucose levels better than the
control rats. This model mimics the transplantation of islet cells in humans and shows
that the addition of the HGF gene greatly enhances the islet cells' function and survival.
In May 2006, a team of scientists orted a breakthrough for gene therapy which they
developed a way to prevent the immune system from rejecting a newly delivered gene.
Delivery of 'normal' gene has been difficult because the immune system the new gene as
foreign and rejects the cells carrying it. This problem was overcome utilizing a newly
uncovered network of genes regulated by molecules known as microRNAs this natural
function of microRNA is to selectively turn off the identity of the at multiple insertion
sites. These multiple ilencing of the transgene in subsequent progeny (Gan, 1989; Yao
Applications of Gene Therapy

Prospects of Gene Therapy

There are a lot of prospects pertaining to gene therapy. Gene therapy as we all know it
is not a ‘cure all’ option. It is not always successful but it means that a disease can be
eradicated for aperson and their future offspring, so it is remedied in not just one
generation but also in subsequent generations (Arena et al., 2011). Gene therapy also
has the potential to ‘silence’ a gene as in the case of a subject with HIV, which had not
yet developed into AIDS, scientists could save them the pain and suffering of the disease
by using gene therapy to ‘silence’ the disease before its onset. I believe that if the cynics
and those who are skeptical to this technique were ever faced with cancer or a child
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born with a genetic disease, they would change their views.

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Conclusion:

Gene therapy is the use of DNA (as a pharmaceutical agent) to treat disease. It is also an
experimental medical treatment that manipulates a gene or genes within cells in order
to produce proteins that change the function of those cells. In gene therapy, DNA that
encodes a therapeutic protein is packaged within a “vector” which is used to get the
DNA inside the cells within the body