GENE THERAPY
INTRODUCTION
The ability to transfect genes into
cells and to cause their expression is leading to the practical emergence of
human gene therapy wherein, functionally active genes are putatively inserted
into the somatic cells of a person requiring the expression of a given protein.
In simple terms, gene
therapy involve insertion of genetic material into a patient’s cells to make
them capable of producing therapeutic protein. Gene therapy paves way to either
replace the missing or defective gene at the origin or arrest undesired gene
expression (viral and oncogene expression) at the origin.
Gene medicines are
generally based on gene expression system that contains a therapeutic gene and
a delivery system. A gene delivery system controls the distribution and access
of a gene expression unit to the target tissue, its recognition by cell-surface
receptors and its intracellular trafficking (Tomlinson and Rollond, 1996).
Gene therapy is the introduction of functional
genetic material into mammalian cells, to replace or supplement the activity of
defective genes. The aim of gene therapy is thus to genetically correct the
defect in major affected organs.
The objectives of
treatment of genetic diseases are to obtain and maintain optimal health, both
in the individual and in the race. The therapeutics approaches exist to restore
normal biochemical relationship: -
(1)
Substrate
Restriction.
(2)
Product Replacement.
(3) Coenzyme Supplementation
(4)
Enzyme Replacement
(5) Other mode of environmental modification involves
pre and
perinatal treatment.
Anderson first hypothesized the idea of human gene therapy
in the late sixties. Gene transfer with retroviruses was made in animals in
eighties. The first gene transfers humans, albeit for a marker gene, was made
in 1983. At present more than 65 clinical gene transfer protocols have been
approve. Simultaneously in the Human Genome Project, a co-ordinate effort is
underway to construct maps of the human genome, including the identification
and localization of all genes. This will useful for the early diagnosis and
treatment of patients with genetic disorders.
APPROACHES FOR GENE THERAPY
Various approaches have
been tried for effective transfer of genes to appropriate target site.
1. Gene modification
a. Replacement therapy: a defective gene is inserted in the genome
b. Corrective gene therapy: replacement of mutant gene with a normal
sequence.
2. Gene transfer in specific cell lines
a. Somatic gene therapy: it involves the insertion of gene into
specific somatic cells.
b. Germline gene therapy: injection of genes into germ cells.
3 Eugenic approach (gene insertion): inserting genes to alter or
improve complex traits of a person.
4.Gene transfer: -
Gene can be introduced into the cells by physical, chemical and
biological methods, for introducing the genes into mammalian cells viral
vectors offer the most promising gene delivery system studied today.
Table 1: Method of Gene Transfer
S no. Method Advantage Disadvantage
1. Physical
(a.)
Microinjection
-------
One cell can be
injected
(b)
Electroporation Simple and
technically ------
(c) Gene gun Simple and technically Limited depth of the
Ideal
for gene mediated DNA penetration
Immunization.
(d) Direct parenteral No inflammatory ------
injection of un- response seen
Complexed DNA
2.
Chemial
(a) Liposomes Gene is targeted to Levels of gene
an
intracellus location,
transferred by
non-toxic immunogenic. are lower.
(b) Ligand mediated cell specific gene transfer Efficiency of . gene delivery
non-immunogenic. transfection low
S no. Method Advantage Disadvantage
3. Biological
Viral vectors
(a) Retrovirus Long terms expressions, Limited to dividing
lack immunogenic proteins, cells.mutafenesis,
no
pre-existing hosts
tumorigenic potentia
immunity.
(b) Adenovirus High levels of gene transfer Short duration of
and
transgene expression. transgene expresion
immunogenic.
(c) Adeno
associated Efficient intergration
into Difficulty in produc-
viruses host genome, association ing the virus in large
with
human disease, non quantity
immunogenic, for periods.
(d) Vaccinia virus High yields of virus achieve Elicits a host
easily,
large DNA inserts immune response.
Possibility of recombination
to produce new strains.
.
(e) Herpes High titre stocks
obtaines Elicits a potent
simplex virus
immuneresponse.
S no. Method
Advantage Disadvantage
4. Other viral
vectors
Include: HIV,
hepatitis B, ------- -------
Infulenza-Virus,
Ebstein Barr,
Cytomegalo
(a) Mamalian
artifical Reduced risk
of Technically
mutagenesis.
difficult.
(b) Neo organ
implant Specific targeting
of Short lasting
cells, of the utility in
enzyme replacement
therapy.
DIABETES
MELLITUS (TYPES AND CAUSES)
The
normal blood glucose level in human’s ranges between 70-90 mg per
100ml.Pancreas contains about a million islets of Langerhans, which constitute
pancreatic tissue, having the cells:
[2,3]
(i) a
- Cells (in outer cortex)-they secrete glucagons.
(ii) b
- Cells (in medulla)-they secrete
insulin.
(iii)
D-or d
- Cells –they secrete somatostatin.
(iv)
PP or F cells- they release of pancreatic polypeptide.
Hyperglycemia
is characterized by more than normal concentration of the blood sugar and
hypoglycemia develops when the blood sugar level falls below the normal range.
The term ‘Diabetes’ to mean large urine volume, the adjective ‘mellitus’ a meaning
of honey.
It is metabolic disorder
characterized by hyperglycemia. Glycosuria, polyurea, polyphagia, hyperlipemia,
negative nitrogen balance and sometimes ketonemia, A wide-spread Pathological
change is thickening of capillary basement membrane, increase in vessel wall
matrix and cellular proliferation resulting in vascular complications, like;
lumen narrowing, early athrerosclerosis, seclerosis of glomerular capillareies.
Retinopathy, neuropathy, peripheral vascular insufficiency, predisposition to gangrene,
and kidney damage.
Two major types of
diabetes mellitus are:
Type
I: - Insulin dependent diabetes mellitus (IDDM), juvenile onset diabetes
mellitus: Probably an autoimmune disorder (a viral etiology is also proposed;
encephlomyocarditis)- antibodies that destroy b-cells of islets of Langerhans in pancreas are often
detectable in blood: insulin in circulation is low or absent: more prone to
ketosis. This type is less common and has a low degree of genetic
predisposition.
Type II: - Non-insulin dependent diabetes mellitus (NIDDM).
Matuty onset diabetes mellitus. There is no loss or moderate reduction in cell
mass: insulin in circulation is low, normal or even high, no anti b-cell antibody is demonstrable: has a high degree of
genetic predisposition: generally has a late onset (past middle age).
Type III: - Others
(A) Genetic defect of cells function
1. Chromosome 12, HNF-1a(MODY3)
2. Chromosome 7, glucokinase (MODY2)
3. Chromosome 7, HNF-4a(MODY1)
4. Mitochondrial DNA
Genetic defects of cells function of diabetic are frequently
characterized by onset of hyperglycemia at early age (before 25yrs.) it
referred as maturity onset diabetes of young (MODY) and impaired insulin
secretion with minimum or no defect in insulin action.
Abnormalities at three genetic loci on different chromosome, the
most common form is associated with mutations on chromosome12 in a hepatic
transcription factor referred to as a hepatocyte nuclear factor (HNF1a).
A second form is associated with mutation in glucokinase gene on
chromosome 7p and result in defective glucokinase molecule. Third form is
associated with mutations in the HNF-4a gene on chromosome 20q HNF 4a is a transcription factor involved in the regulation of
expression of HNF 1a point mutation in mitochondrial DNA have been formed to be
associated with diabetes and deafness.
(B) Genetic defect in insulin action
1. Type A insulin
resistance
2. Leprechaunism
3. Rabson-menden half
syndrome
4. Lipoatrophic diabetes
There are unusual causes of diabetes that result from genetically
determined abnormalities of insulin action; the metabolic abnormalities
associated with mutation of insulin receptor may range from hyper insulinemia
and modest hyperglycemia to severe diabetes. This syndrome was termed type A insulin
resistance.
Leprechaunism and Rabson-menden half syndrome are two-pedriatric
syndrome that have mutation in insulin receptor gene with subsequent alteration
in insulin receptor function and extreme insulin resistance.
(C) Disease of exocrine pancreases
(D) Drug –chemicals and vector associated diabetes .
Insulin
:- It is a two chain polypeptids having 51
AAs MW about 6000, The A chain has 21 while B-chain has 30 AA. The A & B
chain are held together by two sulfide bond . [3]
Synthesis: - It is synthesized in the b-cell of pancreatic islet as single chain peptide preproinsulin (100 AAs) from which 24 AAs are removed to produle proinsulin. The connecting ® C-peptideds (30 AA) split of by proteolysis in gogi apparatus and both stored in granules within the cell.
Preproinsulin (110 AA) ® proinsulin (86 AA) ® Insulin + Cpeptide
Release: - Normally the insulin release 1-2 mg per day and 20mcg per hour on stimulus.
1. Chemically – AAs, fattyacids, glulose, ketonebodies® Activation of glucoreceptor on b cell (chemical signal –incretins)
2.Hormanally – GH, corticosteroids,thyroxine,
Mechanism action: - Insulin act on specific receptor on cell membrane of all cell (mainly liver and fat cells). Insulin receptor has 2 a & 2 b-subunits. Insulin bind to a-subunit & activate b-tyrosine proteinkinase activity. Insulin stimulate glucose transport across cell membrane by ATP dependent translocation of glucose transporter.
Insulin+a-subunit
insulin receptor®internalized
receptor complex®b-tyrosine
residue®phosphorylation-cascade
and sec. Messenger®
glucose transport across cell membrane (ATP dependent)
Action: -
1.
Facilitate – glucose transport across cell
membrane.
2.
Insulin enhance intracellular utilization
of glucose ( phosphorylation form glulose-6-phosphate ) by increasing
glucokinase.
3.
Facilitate glycogen synthesis from glucose
in liver, muscle & fat cells stimulating glycogen synthesis glycogenolysis
by inhibiting phosphorylase.
4.
Inhibit gluconeogenesis ( from protein )
5.
Inhibit lipolysis in adipose tissue.
TREATMENT
There are diverse strategies for gene therapy of diabetes
mellitus. Prevention of cell autoimmunity is a specific gene therapy for
prevention of type 1 (insulin-dependent) diabetes in a preclinical stage,
whereas improvement in insulin sensitivity of peripheral tissues is a specific
gene therapy for type 2 (non-insulin-dependent) diabetes. Suppression of -cell
apoptosis, recovery from insulin deficiency, and relief of diabetic
complications are common therapeutic approaches to both types of diabetes.
Several approaches to insulin replacement by gene therapy are
currently employed:
1) Stimulation of -cell growth,
2) Induction of -cell differentiation and regeneration,
3) Genetic engineering of non-cells to produce insulin, and
4) Transplantation of engineered islets or cells.
5) Synthesis of insulin.
In type 1diabetes, the therapeutic effect of -cell proliferation
and regeneration is limited as long as the autoimmune destruction of cells
continues
Therefore, the utilization of engineered non-cells free from
autoimmunity and islet transplantation with immunological barriers are
considered potential therapies for type 1 diabetes. Proliferation of the
patients' own cells and differentiation of the patients' own non-cells to cells
are desirable strategies for gene therapy of type 2 diabetes because
immunological problems can be circumvented.
At present, however, these strategies are
technically difficult, and transplantation of engineered cells or islets with
immunological barriers is also a potential gene therapy for type 2 diabetes.
In 1990, the first full-scale gene therapy was
applied to the treatment of adenosine deaminasedeficiency in the USA.Since
then, more than 4000patients have been treated by over 400 gene therapy
protocols worldwide. About two-thirds of the target diseases of gene therapy
are malignantneoplasms, followed by AIDS, hereditary monogenic diseases, and
vascular diseases. Genetherapy has not been applied to the treatment of
diabetic patients, but the possibility of its clinical application has been
pursued using diabetic animal models.
There are diverse
approaches to gene therapy for diabetes mellitus [Table II], and the problems inherent in these approaches that
still need to be solved as well as future prospects are discussed here.
Table II. Gene therapy strategies for diabetes mellitus
Therapeutic Approaches Types of
Diabetes
I. Protection of Pancreatic Cells
(1) Prevention of -cell autoimmunity Type 1
(2) Suppression of -cell apoptosis Types 1 and 2
II. Recovery from Insulin Deficiency
(1) Stimulation of -cell proliferation Mainly type 2
(2) Insulin secretion from non-cells Types 1 and 2
(3) Differentiation of non-cells into
Cells and -cell regeneration Mainly
type 2
(4) Islet transplantation
Types 1 and 2
III. Improvement of Insulin Sensitivity Type 2
IV. Gene Therapy for Monogenic
Diabetes Mellitus
Monogenic
V. Gene Therapy for Diabetic
Complications
Types 1 and 2
VI. Synthesis of insulin. Types 1
I. PROTECTION OF PANCREATIC CELLS
(1) Prevention of -Cell Autoimmunity [12-16]
Type 1 diabetes mellitus
(insulin-dependent diabetes mellitus) results from an autoimmune destruction of
cells by T lymphocytes. In most cases, cells are gradually destroyed before the
clinical onset of type 1 diabetes mellitus, and serum autoantibodies for cell
antigens and lymphocytic infiltration of pancreatic islets are detected in the
preclinical stage of the disease. At this stage, modifications of the immune
system or cells by gene therapy are attempted to prevent the autoimmune
destruction of cells.
When
transforming growth factor-1 (TGF-1), an Immunosuppressive cytokine, was
systemically expressed in nonobese (NOD) mice, an animal model of type 1
diabetes, by intramuscular injection of a plasmid containing this gene, these
mice were protected from insulitis and diabetes, but this protection was
associated with a considerable elevation of the plasma TGF-1 level. However,
systemic immunity was affected with
excessiveTGF-1, and adverse effects were feared.
The transgenic expression of I-E, an MHC class II antigen that
protects against type 1 diabetes, in NOD mice can completely prevent the
development of autoimmune insulitis. However, gene transfer into the whole body
or the entireimmune system is difficult. Therefore, realistic target cells for
gene transfer are -cell-specificcytotoxic T cells or cells them selves.
The transgenic expression of immunosuppressive peptides, such as
interleukin-4 (IL-4), TGF-1, or calcitonin gene-related peptide, in islet cells
of NOD mice prevents the onset of type 1 diabetes. Furthermore, islet-specific
cytotoxic T cells can be used as a vector for the local expression of transgenes
in pancreatic islets as follows: after If the expression of MHC antigens in
cells is suppressed by gene transfer of adenoviral E3gene into cells,
autoimmune diabetes may be prevented. However, an effective method for gene
transfer into cells in vivo has not been developed. Injection of
adenoviral vector into the pancreatic artery of an isolated human pancreas ex
vivo has been reported to result in transduction of approximately 50% of
the cells. Gene transfer into the pancreas using the adenoviral vector can also
be achieved by direct pancreatic injection or retrograde delivery of adenovirus
into the pancreatic duct.
However, as long as the adenoviral vector is used, acute
pancreatitis is also induced. Therefore, an inflammation-inducible vector such
as the adenovirus is not suitable for gene transfer into the pancreas in
vivo. Lastly, to prevent autoimmune -cell destruction by gene therapy, a
method for selection of appropriate subjects for gene therapy must be
established. It is important to detect subjects who will develop type 1diabetes
in the future, in the period where sufficient -cell mass remains before the
onset of the disease. For prediction of type 1 diabetes, combined autoantibody
screening collecting the islet-specific T cells from the pancreas of NOD mice,
IL-10, an immunosuppressive cytokine, was over expressed in these cells using a
retroviral expression vector, and these transfectants were injected in NOD
mice. The administered T-cell transfectants accumulated around the pancreatic
islets, secreted IL-10, and protected
cells from autoimmune destruction. Furthermore, the T-cell transfectants
were not cytotoxic to cells [Fig. (1)].
Collection
of Islet
|
|
Secretion
HOG MOUSE
Islet antigen
Antigen Presentative
Cell
IL-2
T-cell
prolliferation
IL10
IL10
IL10
IL10 IL10
 |
|||
 |
|||
IL-10 gene transfer by retroviral vector
 |
IL10 IL10 IL10
Selection of T-cell transfectant
IL10
IL10
 |
Accumulation
of transfectant
around islet
|
Administration
of transfectants into NOD MICE
Figure 1.A gene therapy model for type 1
diabetes using islet autoantigen-specific T lymphocytes.
Including a quantifiable measure of glutamic acid decarboxylase
has been shown in families and in populations.
(2) Suppression of -Cell Apoptosis [17-19]
The decrease in the number of pancreatic cells is a common feature
of type 1 and type 2(non-insulin-dependent) diabetes mellitus, and is due to
apoptosis of cells. Transfection of an anti-Fas ribozyme into mouse insulinoma
cells inhibits Fas-mediated apoptosis. When the anti-apoptotic protein Bcl-2 is
expressed in mouse and human cells in vitro using herpes simplex viral
(HSV) vector, Bcl-2-transfected cells become resistant to apoptosis induced by
cytotoxic cytokines such as IL-1, tumor necrosis factor-, and interferon.
The HSV vector may be clinically applicable, because human cells
can effectively be transfected by this vector. However, suppression of-cell
apoptosis may also promote insulinoma development. Pancreatic islet cells are
terminally differentiated and refractory to stable transfection by retroviral
vectors, which require the breakdown of the nuclear membrane during cell
division in order to insert the transgene into the host cell genome. On the
other hand, human immunodeficiency virus type 1 (HIV-1)-based lentiviral
vectors can transduce dividing and non- dividing cells.
Furthermore, the transduced tissues demonstrate the long-term
expression of transgenes. Originally, HIV-1 can infect only a narrow range of
tissues and cell types including CD4-positive T lymphocytes. In contrast, the
HIV-based lentiviral vectors are pseudotyped with vesicular stomatitis virus G
glycoprotein (VSV-G), and can transduce a broad range of tissues and cell
types, because VSV-G binds to phosphatidylserine of the cell membrane. Thus,
the HIV-based lentiviral vectors have recently been applied to gene transfer
into cells.
II. RECOVERY FORM INSULIN DEFICIENCY
(1) Stimulation of -Cell Proliferation [20-22]
In diabetic patients, -cell function is impaired by cytokines,
glucose toxicity, lipotoxicity, oxidant-stress, etc. In islets of diabetic
Goto-Kakizaki (GK) rats, decreased expression of the proteins necessary for
insulin exocytosis is in part responsible for impaired -cell function, and the
over expression of these proteins in GK rat islets by adenovirus-mediated gene
transduction increases glucose-stimulated insulin secretion.
However, a gene therapy
strategy for recovery of -cell function has hardly been considered, because
there is no effective approach in the presence of a decreased
number of cells in diabetic patients. Therefore, we are interested in
developing a gene therapy strategy for stimulation of –cell proliferation.
The proliferation of cells is stimulated by
various secreted proteins, including insulin-like growth
factors (IGFs)-I and II, platelet-derived growth factor, growth hormone (GH),
prolactin (PRL), and placental lactogen. In rats bearing GH- and PRL-producing
tumors, GH-expressing transgenic mice, and pregnant rats, the -cell mass
increases and insulin secretion is enhanced, whereas in pituitary GH- and
PRL-deficient dwarf mice, islet volume decreases 2-5 times. Recently,
transgenic over expression of parathyroid hormone-related protein or hepatocyte
growth factor (HGF) in cells was also reported to double or triple the islet
mass. In regard to-cell signal transduction, insulin receptor-substrate-2
(IRS-2), insulin receptor-related receptor (IRR), and cyclin-dependent kinase 4
(Cdk4) are thought to play important roles in -cell proliferation. IRS-2
mediates insulin/IGF signaling, and IRS-2-null mice show a decrease in islet
mass.
IRR
is an orphan receptor, highly expressed in islet cells, and stimulates tyrosine
phosphorylation of IRS-1 and IRS-2. Cdk4-null mice develop insulin-deficient
diabetes due to a reduction in cells, whereas "knock-in" mice
expressing a mutant Cdk4 that cannot bind its inhibitor protein display -cell
hyperplasia. These factors may be used for-cell proliferation by gene therapy
in the future.
(2) Insulin Secretion from Non-Cells [23-25]
Because islet-specific antigens are not expressed
in non-cells, genetically engineered non-cells easily escape from autoimmune
destruction in type1 diabetic patients. Moreover, the utilization of
insulin-secreting non-cells can solve the problem of an insufficient supply of
islets for transplantation. Fibroblasts can be easily obtained from a diabetic
patient, and can be returned after gene transfer to the same patient without
immunological rejection.
However,
the fibroblasts transduced with proinsulin gene cannot remove C-peptide from proinsulin,
because proprotein convertases1 and 2 are not expressed in the fibroblasts.
Furthermore, in fibroblasts, translated proinsulins are not stored in secretory
granules, but are secreted at a constant rate independent of glucose
concentration, i.e. constitutive secretion. Processed bioactive insulin
can be produced by non-cells, when the amino acid sequences at the cleavage
sites of proinsulin are mutated to tetrabasic recognition sites for furin,
another proprotein convertase [Fig. (2)], because furin is ubiquitously
expressed in most cells.
When endocrine cells such as At T20 pituitary
cells are used for the expression of the proinsulin gene, normal processing of
proinsulin and insulin storage in secretory granules are observed, but insulin
is not secreted in response to high glucose concentration, as in the case of
other non-cells.
A number of attempts have been made to achieve
glucose-dependent insulin secretion from non-cells. Because hepatocytes respond
to glucose and insulin concentrations and regulate the expression of various
metabolic enzymes, they are considered to be the most suitable non-cells for
glucose-dependent insulin secretion.
If insulin is expressed in hepatocytes under the
control of the L-type pyruvate kinase gene promoter, a glucose-dependent
promoter, insulin gene transcription will be activated by glucose [Fig.
(3)]. However, in cells, an intense response of insulin secretion to
glucose is primarily regulated at the level of exocytosis of insulin-containing
granules.
C-PEPTIDE
 |
Gly-val-glu -Asp-Pro-glu-val–ala-Arg-glu
Arg- Arg
Lys-Arg
B-Chain B-Chain
- - Pro-Lys-Ser gly-lue-val - -
- - Pro-Lys-Ser gly-lue-val- -
B-Cha A-Chain
(Furin
cleavage site)
Arg- Arg
Lys-Arg
Lys-Arg-glu-Asp-Pro-glu-val-Ala-Arg
Mutated C-peptide-Furin
cleavalge site
Figure 2.Amino acid sequences of normal human proinsulin and its
mutant containing furin cleavage sites at the A-chain/C-peptide and
B-chain/C-peptide junctions. A furin recognition motif for peptide cleavage is
Arg-Xaa-Lys/Arg-Arg.
Increase blood glucose level increase
insulin secretion
Decrease blood insulin level decrease blood
glucose
Increase blood insulin
Increase insulin
secretion
 |
Figure 3.Regulated insulin secretion from
genetically engineered hepatocytes.
Therefore, the regulation of insulin secretion
only by glucose-dependent gene transcription insufficient for tight glycemic
control. Moreover, if insulin secretion is regulated only at the transcription
level, insulin translation from its mRNA continues after glucose-dependent
transcription termination, increasing the risk of hypoglycemia.
Thus, the mechanisms preventing excessive. Insulin secretions have
also been considered. When insulin is expressed in hepatocytes under the
control of the P-enolpyruvate carboxykinase promoter, which is inactivated by
insulin, excessive insulin secretion is prevented. Hypoglycemia-induced
glucocorticoid secretion has also been used as a negative-feedback mechanism of
insulin production; i.e., a glucocorticoid-responsive promoter was
introduced in the 3' region of insulin cDNA in reverse orientation so that
antisense insulin mRNA is produced in response to glucocorticoids.
Because
insulin secretion from hepatocytes has also been successful in in vivo
studies using retroviral and adenoassociated viral vectors, hepatocytes appear
to be the most promising candidate for insulin-producing non-cells in clinical
application, at present. The liver is the target organ for insulin, and is exposed
to the highest concentration of insulin secreted from the pancreas into the
portal vein. Therefore, insulin expression in hepatocytes in vivo is conducted
on a near-physiological plane. Although it may be difficult to accomplish a
rapid post-prandial insulin secretion from genetically engineered hepatocytes,
The stimulation of -cell proliferation by gene transfer is considered primarily
for treatment of type 2 diabetes, because its effect is limited in type
1diabetic patients as long as autoimmune destruction of cells persists.
The prevention of ketoacidosis in type 1 diabetic patients by
sustained insulin expression from the liver can be anticipated. In view of
achieving sustained insulin secretion, it is easier to express insulin in
skeletal muscles by direct injection of insulin expression vectors including
naked plasmid and viral vectors. For a steep response of insulin secretion from
non-cells, a method for storing insulin in the endoplasmic reticulum (ER),
instead of in secretory granules, was recently developed. In this method,
insulin is expressed as a fusion protein with a conditional aggregation domain
that interacts with itself in a ligand- reversible manner.
Aggregates of insulin fusion proteins accumulate in the ER.
Addition of a cell-permeant ligand dissolves the aggregates, and the fusion
protein exits the ER and is secreted through the constitutive secretory
pathway. The aggregation domain and C-peptide are removed by furin in the
trans-Golgi apparatus, resulting in bioactive insulin.
In cultured hepatocytes or pituitary cells, the co-expression of
insulin and glucose transporter-2 (GLUT-2) or glucokinase improves the insulin
secretion response to glucose. However, when cultured cells are transplanted
into diabetic patients, there are many difficult problems including the
protection of transplanted cultured cells from the host immune system, the
control of the proliferation of these cells, and gradual changes of their
phenotypes such as decreasing insulin secretion. Even when the patient's own
fibroblasts transduced ex vivo are again transplanted into the same
patient, the stability of transgene expression is not guaranteed, i.e.
the transgene expression level often decreases gradually.
As mentioned above, it is difficult to mimic the
normal response of insulin secretion to glucose in non-cells. Therefore, the
interest in creatingimitation cells from non-cells is decreasing, and other
gene therapy strategies for diabetes mellitus are considered to be more
promising.
(3) Differentiation of Non-Cells into Cells
and-Cell Regeneration [26-28]
Recent developmental studies revealed that epithelial cells of the
pancreatic duct differentiate into islet cells. The earlier hypothesis had
implied that highly differentiated endocrine cells do not proliferate or
regenerate, like nerve cells. Indeed, the ability of mature endocrine cells to
proliferate seems to be limited, but even in the adult pancreas, a considerable
number of endocrine cells can regenerate from the epithelial cells of
pancreatic ducts after islet cells have been damaged. Especially after a
partial pancreatectomy, vigorous islet neogenesis is observed. Therefore, if
the mechanism of islet neogenesis is clarified, cells in diabetic patients may
be regenerated by gene therapy. A number of protein factors are involved in
-cell differentiation and regeneration. In particular, the regenerating protein
(Reg) family including Reg I and is let neogenesis-associated protein is
thought to play an important role in -cell regeneration. However, there are
also some problems to overcome in this strategy.
Firstly, at present, our knowledge of islet development and
neogenesis is too scant to develop gene therapy strategies for inducing is let
neogenesis. Secondly, it is considered that the mechanism of development of
fetal islets differs from that of islet neogenesis in adult pancreas, so that
developmental findings in fetal islets may be inapplicable to islet
regeneration therapy in the adult. Thirdly, even though islet neogenesis can be
induced, there is a limited therapeutic effect on type 1 diabetes, as long as
the autoimmune destruction of cells continues. Even in type 2 diabetes, cells
may be unsuccessfully regenerated in the presence of glucose toxicity and
lipotoxicity against cells. Lastly, rapidly regenerated cells are probably less
functional than normal cells. Nevertheless, gene transfer into epithelial cells
of the pancreatic ducts can be achieved by in vivo retrograde injection
into the pancreatic ducts, and islet regeneration from the pancreatic ducts is
a potential strategy for future gene therapy.
In addition to cells of
the pancreatic duct, hepatocytes are also a candidate for non-cell
fordiffentiation in to cells and hepatocytes are derived from primitive
duodenum and have certain developmental similarities such as
the common expression of
hepatocyte nuclear factors (HNFs) in both cell types. Recently, it was reported
that gene transfer of pancreatic and duodenal homeobox gene 1 (Pdx1) into
hepocyte by adenoviral vectors induces transdifferentiation of hepatocytes into
insulin-secreting cells, and ameliorates streptozotocin (STZ)-induced
hyperglycemia in mice. Because Pdx1 is an essential factor for–cell
differentiation, it may also be used in gene therapy to induce the transdifferentiation of other endoderm-derived cells
into cells.
(4) Islet Transplantation [29-30]
Excellent therapeutic results of allergenic islet transplantation
have recently been reported in seven patients with type 1 diabetes mellitus by
means of a percutaneous transhepatic portal embolization of islet transplants.
However, to establish islet transplantation as a standard therapy for diabetes
mellitus, it is necessary to ensure a supply source of islets. In xenogeneic
islet transplantation, the islet supply is potentially unlimited. Although
transplantation studies using porcine islets are most advanced, immunological
rejection is still the major problem. To prevent rejection of transplanted
islets, immuno isolation and local immune suppression are considered.
The principle of immunoisolation is to separate transplanted
islets from the host immune system by a selectively permeable membrane, thus
eliminating the need for immunosuppressive drugs. Low-molecular-weight
substances such as nutrients, electrolytes, oxygen, and biotherapeutic
products, i.e. insulin, are exchanged across the membrane, while
immunocytes and antibodies are excluded. However, it is difficult to achieve
complete protection from immunological rejection, because the insulin-permeable
pores of the membrane also allow the efflux of small antigens and the influx of
complement components and inflammatory cytokines. Especially in type 1
diabetes, insulin itself, which is secreted from encapsulated islet grafts, is
an autoantigen recognized by autoimmune cytotoxic T cells.
Moreover, an oxygen supply is crucial for the proper functioning
and long-term survival of the islet graft. However, improving blood vessel
formation, around an immunobarrier membrane induces T cell recruitment,
resulting in cell apoptosis by membrane-permeable cytokines. For local immune
suppression immunosuppressive proteins are expressed in transplanted islets by
gene transfer, i.e. a cell- based therapy for diabetes mellitus
including islet transplantation has recently been combined with gene therapy [Fig.
(4,A,B)].
When islets are transplanted with Fas ligand-expressing cells,
immunological rejection of the islets is prevented. Fasligand binds to Fas of T
lymphocytes, and is thought to induce their apoptosis and to protect the islet
graft. Paradoxically, Fas ligand expression in the islets themselves by gene
transfer accelerates insulitis and islet destruction. As shown in this example,
the immune response is complicated, and differences in species or experimental
models often lead to conflicting results.
Therefore, when the immune system is modulated by gene therapy,
the complexity of immune regulation should be considered. The pig has been
identified as the most suitable animal donor. The antigenicity of pig organs is
reduced by transgenic approaches. Recent advances in animal cloning might
accelerate the pace of these efforts. In addition to graft rejection, the risk
of xenobiotic viruses is another problem of xenotransplantation.
IMMUNO-ISOLATION
 |
Express
Immunosuppressive Induction of
Insulin
Protein Tolerance
|
|||
 |
Decrease
antigenicity
 |
X
Transplantation
Augumentation
Suppression Growth Safety
Of b cell of apoptosis control system
Function
(tetracycline (thymidine
(Pd --, glucokinase responsive kinase)
Glu
T2, Kin G2,
Sur 1 UG1, PC etc)
Figure 4. (B) Transplantation of genetically
engineered cells or islets. Various steps for gene transfer are shown. "Augmentation
of -cell function" is a gene therapy strategy to restore rapid insulin
secretion in response to glucose in low-differentiated cells after long-term
culture.
All species, including humans and pigs, carry endogenous
retroviruses. Pig endogenous retroviruses (PERV) can infect human cells in
vitro and mice in vivo after pig islet xenotransplantation.
Because immunosuppressive drugs are used in transplanted patients, PERV
infection may induce serious disease. Even though the PERV sequence could be specifically
inactivated or removed from the genome, the elimination of all viruses is
difficult. However, PERV infection has not been detected by PCR analysis in any
of the patients who have been treated with various living pig tissues.
In allergenic transplantation, the problems of graft rejection and
retroviral infection can be tackled more easily than in xenotransplantation.
The major issue of allergenic transplantation is the shortage of human donor
islets. The amount of adult cells obtained from cadaveric pancreases is
insufficient for the demand. In consideration of social issues, the extensive
use of human fetal islets is unlikely. However, because the fetal islets are
rich in-cell precursors, a sufficient islet supply is ensured if these precursor
cells can proliferate and differentiate into mature cells.
For proliferation and
differentiation of human adult and fetal islet cells in vitro,
extracellular matrix and HGFare used. In general, however, as cells
proliferate, their differentiated phenotypes including cells that secrete
insulin gradually decrease. Also when cultured human -cell lines are expanded in
vitrofortransplantation, a similar problem occurs: cell lines growing at
high growth rates show decreased insulin secretion. A better understanding of
the mechanisms of-cell proliferation and differentiation may
enable the proliferation of highly differentiated cells and
re-differentiation of low-differentiated cells after proliferation, by the
addition of -cell differentiation factors such as nicotinamide or by gene transfer.
When cultured -cell lines
are used for transplantation, it is important to control the number of these
cells, in addition to the maintenance of differentiated -cell function;
otherwise the –cell lines grow infinitely, and the resultant tumors and hypoglycemia
are life-threatening. When an oncogene is expressed in transplanted -cells
under the control of a tetracycline-responsive promoter, the number of these
cells can be controlled in vivo by the administration of tetracycline.
In the case when transplanted cells form tumors, two safety systems for graft
removal are proposed. One is a method using an antibody against a specific
antigen of the transplanted cells. The other is a method using a suicide vector
encoding thymidine kinase gene, which induces apoptosis of transfectants in the
presence of gancyclovir or acyclovir.
However, these systems do
not guarantee complete safety. In addition to the decreasing insulin expression
in transduced non-cells, the expression level of genes that function as the
safety system often also decreases gradually. Insulin-secreting
cells derived from embryonic stem (ES) cells can be used for –cell
transplantation. The expression of the neomycin-resistant gene under the
control of the insulin promoter in ES cells enables us to select
insulin-secreting cells by G418, a derivative of neomycin, among differentiated
ES cells in culture without leukemia inhibitory factor (LIF) [Fig. (5)].
If custom-made ES cells of the diabetic patient himself are used for
transplantation, the patient is free from long-term immunosuppressive therapy.
Although nuclear transfer of a patient’s somatic cell nuclei to human
fertilized eggs can obtain ES cells of individual patients, this protocol is,
at present, regulated by law in many countries.
Generally, islet transplantation results in better glycemic
control than -cell transplantation, because glucagons secreted from pancreatic
cells plays an important role in glycemic control, as well as insulin from
cells. Indeed, in HNF3-null mice, a 70% reduction in pancreatic proglucagon
gene expression induces hypoglycemia, leading to postnatal death. Therefore, an
unlimited supply of whole islets is desirable for transplantation. Recently, it
was reported that whole islets can be generated in vitro from cultured
epithelial cells of the pancreatic duct, i.e. islet progenitor cells.
Using this method, the number of available islets per pancreas can be increased
by nearly 104.
Insulin promoter Neomycin Phosphoglycerate Hygromycin
Resistant
kinase promoter resitant gene
Gene
Gene transfer Addition of
hygromycin
 |
Selection of tranfected ES cell Removal of LTF
 |
Induction of differentiation
Addition of G-418
Selection of Insulin Addition
of nicotinamide
Secreting cell
Highly differentiate b cell
Figure 5. Harvest of insulin-secreting
cells derived from ES cells. ES cell transfectants can be selected by
hygromycin, because the phosphoglycerate kinase promoter expresses the
hygromycin-resistant gene in ES cells. After the induction of differentiation
by removal of LIF, insulin-secreting cells are selected by G418, because in
these cells, the insulin promoter expresses the neomycin-resistant gene. For
final differentiation and maturation, the insulin-secreting cells are cultured
with nicotinamide, a -cell
differentiation factor.
III. IMPROVEMENT OF INSULIN SENSITIVITY [31-34]
Protection of cells from autoimmune destruction is a gene therapy
strategy specific to type 1 diabetes mellitus, whereas improvement of insulin
sensitivity is a strategy specific to type 2 diabetes mellitus. The major
insulin target organs are the liver, skeletal muscles, and adipose tissues, and
a number of protocols to improve insulin sensitivity are conceivable. However,
animal experiments using this strategy are few in number at present. Because
the P-enolpyruvate carboxylase gene promoter is activated by a decrease in
intracellular insulin signaling, this promoter can be used for transgene
expression in the liver under conditions of insulin resistance. In transgenic
mice expressing glucokinase (which is the rate-limiting enzyme in glycolysis
and is present in the liver due to the P-enolpyruvate carboxylase gene
promoter), glycolysis is induced while gluconeogenesis and ketogenesis are
blocked even after STZ-induced diabetes. In type 2 diabetic patients, the
expression of GLUT-4 decreases in skeletal muscles.
Therefore, gene transfer of GLUT-4 to the skeletal muscles of
diabetic patients is thought to improve their insulin sensitivity. Gene therapy
with leptin had been expected to ameliorate obesity and hyperglycemia by
insulin resistance. Transgenic expression of leptin in leptin- deficient ob/ob
mice results in dramatic reductions in food intake and body weight, as well as
the normalization of serum insulin levels and glucose tolerance. Even in normal
rats, leptin over expression induces dramatic effects including adipocyte
transformation from cells that store triglycerides to fatty acid-oxidizing
cells, disappearance of body fat, and decreases in plasma triglycerides and
insulin levels. Because massive production of free fatty acids from
Insulin-resistant adipose tissue results in -cell apoptosis, improvement of
insulin sensitivity by leptin is favorable for protecting -cells from
apoptosis.
However, the conclusion of the first clinical trial in humans
using recombinant leptin is not very impressive as a potential weight-loss
drug. Some obese volunteers given leptin lost more weight than controls. The
differences were statistically significant, but only in obese subjects given
the two highest leptin doses. Although lepton did cure the obesity of a
youngster found to have defects in his leptin gene, such mutations are rare.
IV. GENE THERAPY FOR MONOGENIC DIABETES MELLITUS [35-38]
Diabetes mellitus is sometimes caused by mutations of single genes
including insulin, insulin receptor, glucokinase, Pdx1, HNF1, HNF1, HNF4, and
mitochondrial genes. However monogenic mutations are found in only less than,
5% of diabetic patients, and gene therapy for monogenic diabetes has not been
planned at present. As a gene therapy strategy of monogenic diseases, a
supplement of normal protein by the expression of the normal gene has been
considered previously, rather than mutation repair. However, in vivo
site-directed mutagenesis has recently been developed using chimeric RNA/DNA
oligonucleotides.
This method has been
applied to repair of point mutations in several monogenic diseases including
sickle cell anemia, albinism, Crigler-Najjar syndrome, hemophilia, and muscular
dystrophy. The detectable level of gene conversion of the mutant allele to the
normal sequence holds promise as a therapeutic method for the treatment of
monogenic diseases.
V.GENE THERAPY FOR DIABETICCOMPLICATIONS [39-42]
Because diabetes mellitus is a metabolic disease and affects
various organs, many gene therapy strategies for diabetic complications are
conceivable. However, gene therapy for vascular complications is emphasized at
present. In particular, atherosclerotic lesions such as coronary heart disease
and foot gangrene have already been treated with gene therapy. Vascular
endothelial growth factor (VEGF) is the most promising factor for angiogenic
therapy of ischemic tissues. By both percutaneous catheter-based arterial
delivery and direct intramuscular injection of naked plasmid DNA gene therapy
with VEGF ameliorates the ischemic hind limb.
In addition, VEGF has been used for therapy of myocardial
ischemia. The naked plasmid DNA or adenoviral vector encoding VEGF is
intraoperatively used for direct intramyocardial administration, leading to
reduced symptoms and improved myocardial perfusion in patients with chronic
myocardial ischemia. Catheter-based transendocardial injection of angiogenic
factors may provide equivalent benefit without the need for surgery. Gene
therapy by VEGF may also rescue diabetes-related impairment of angiogenesis and
neuropathy. However, it should be considered that VEGF has been identified as a
primary initiator of proliferative diabetic retinopathy and a potential
mediator of nonproliferative retinopathy.
In regard to gene therapy for diabetes mellitus, it is difficult,
at present, to accomplish glucose-dependent rapid insulin secretion from non-
cells. Therefore, transplantation of genetically engineered cells or islets
with immunoisolation is expected to be the methods of choice for excellent
glycemic control [Fig. (4,A,B)]. However, gene therapy strategies for
diabetes mellitus do not focus only on "recovery from insulin deficiency".
Along with the technical progress in gene therapy, diabetes mellitus will
certainly be treated by gene therapy.
VI. SYNTHESIS OF HUMAN
INSULIN [1]
Insulin used today by
diabetic patient mostly produced by E.coli bacteria (Eli-Lilly and company’s).
Proinsulin or insulin synthesized by bacteria harboring plasmid that contains
recombinant DNA complementary to mRNA that carries transcript for proinsulin (Fig.6)
The simplest example of
the generation of the novel protein involves the redesigning enzyme structure
by site directed mutagenesis. The approach has been utilized by winter and
associates (winter et.al.1982; Wilkinson et.al. 1984). A detailed knowledge of
structure of enzyme tyrosyl-tRNA synthetase obtained from Bacillus
stearothermophilus helped them to predict point mutations in the gene, which
increased the enzyme affinity for the substrate ATP.
These
changes were introduced and in one case, a single amino acid change could
improve the affinity for ATP by a factor of 100. Furthermore, the approach
helped in increasing the stability of enzyme. Similarly the gene manipulation
coupled with the technique of site-directed mutagenesis was proved by works on
subtilisin. Most of the properties like catalysis, substrate specificity. Ph
and stability to oxidative, thermal and alkaline oxidation inactivation than
was the wild-type enzyme.
ATG
TGA ATG TGA
Synthetic A chain gene Synthetic B chain
gene
(63 nucleotides ) (63 nucleotides)
|
|
|
|
|
|
|
|
|
|
|
|
Figure 6.synthesis of proinsulin by
reconbinant DNA tech .
SAFETY
CONSIDERATIONS
Human gene therapy has
progressed from speculation to reality within a short span. The first clinical
gene transfer (albeit only a marker gene
NeoR/TIL) in an approved protocol was attempted successfully on 22 May, 1989,
at National Institute of Health, Bethesda, MD for malignant melanoma. The first
federally approved gene therapy protocol, for correction of adenosine deaminase
(ADA) deficiency. Began on 14 September 1990.
At National institute of Health Bethesda MD in
spite of wide application they too have problems when brought in practice. For
example; the problems encountered in attempting to correct a single gene
disorder like thalassaemia. With an objective to replace the product of a
defective or missing or b globulin gene with that
of a normal gene .Now the question arises what is our target cell for insertion
of the normal gene? And would the new genes function properly in recipient
cells?
To cure a genetic blood disorder like
thalassaemia, a ‘good’ gene must be inserted into hemopoietic stem cells. The
self-sustaining cell population from which are derived all the formed element
of the blood fire we can’t identify the human stem cells, as they can only be
assayed in murine systems. Secondly till now efficient method is discovered or
available using which successful introduction of corrective gene could be
affected. [1]
Another problem, which
comes across, is the safety in transferring genes into foreign cells. But most
worrying part is the possibility that the, new’ gene might activate an ontogeny
and may give rise to neoplastic change in a particular cell population.
Besides the medical
concerns, there are a number of philosophical, ethical and the logical
concerns. Though there is a general consensus that somatic cell gene therapy
for the purpose of treating a serious disease is an ethical therapeutic option.
Considerable controversy exist as to whether or not germline gene therapy would
be ethical.
Immunological
problems following gene therapy :-
The
major problem of human gene transfer therapy is, regardless of the route of
gene transfer, it elicits an immune response in the recipient. However, there
is very little information available about the antigenic properties of
‘foreign’ proteins of this type. A number of genetic disorders have been
corrected successfully using bone marrow transplantation. But this is always
followed by the administration of drug that suppresses the immune system and
therefore the antigenic properties of the newly introduced protein may well be
masked.
Pre-requisites for human gene therapy
Before the patient is
subjected to gene therapy, several, essential prerequisites are to be
fulfilled. They are
1.
To isolate the appropriate gene and to
define its major regulatory regions.
2.
Identification and harvesting of
appropriate target cells and development of safe and efficient vectors with
which the new gene could be introduced.
3.
Clear evidence of experimental details on
the adequate functioning of the inserted gene, life span of recipient cell and
that no untoward effect exists. Should be ensured.
4.
Last but not the least, the patient or
their family must be fully counseled.
Other clinical use of gene therapy
Diseases wherein gene
therapy has been focused upon include-
¨
Cystic fibrosis
¨
Thalassaemia
¨
Malignant
¨
Pediatric AML
¨
Adenosine deaminase deficiency SCID
¨
Hemophilia B
¨
Chronic myleogenous leudemia
¨
Hepatitis
¨
Hypercholesterolemia
¨
Cardiovascular diseases-Hypertention, Ischaemic heart disease
¨
Acquired immunodeficiency syndrome (AIDS)
¨
Malaria
¨
Influenza
Current Status of Gene Therapy
Gene therapy encompasses
both the replacement of missing or defective genes and augmenting existing
biological processes for fighting disease. Although it was expected that the
first applications of gene therapy would be directed toward correction of
genetic defects, in fact, that has not been the case. The first actual gene
transfer protocol involved patients with cancer. The purpose of this trial was
not therapy, per se, but to see if genetically "marked" cells from a
small group of patients would behave as predicted upon reintroduction into the
patients' blood stream.
The point want to
emphasize is that we are at a very early stage in the development of this
technology. The first targets for gene therapy trials will be determined by the
available scientific capabilities. Some of the thousands of diseases caused by
just the tiniest change in a patient's genetic code may not be treatable using
these methods for quite some time. The reasons for such limitations are varied.
Almost all inherited metabolic disorders are the result of improperly
functioning proteins, especially enzymes. Enzymes are the catalysts that permit
us to extract nutrients from the food we eat, to transfer energy enabling us to
perform tasks, to send signals from one cell to another, and to detoxify and
excrete the end products of these life processes. Enzymes are essential to life
and a defect in the gene coding for these compounds would be lethal to the
developing fetus. Every living organism relies on the appropriate enzymes being
present at the right time and in the right amounts. Therefore, the simple
replacement of a defective gene may not be sufficient to improve the condition
of the patient. Exquisitely fine regulation of production of the enzyme at the
molecular level is also crucial in some cases.
Another example of how
complicated genetic intervention can be is the fact that we may need to reach
the genes in a specific organ like the islets of Langerhans in the case of
patients with diabetes. It may not be feasible or practical to rely on
surgically removing the target cells, altering them in a petridish in the
laboratory and returning them to the patient. Other options include creating
methods that will enable the replacement genetic material to home in on the
target cells or tissues, such as using viral delivery systems that are already
accustomed to reaching the desired cells, or selecting for treatment those
conditions that can be remedied without need for such specificity. Both of
these options are under study.
The
early candidates for gene therapy therefore, are those defects that may be
remedied in a fairly simple fashion by introducing a gene that codes for a
product that does not require careful regulation but can be functional and
useful in any amount while present in the general circulation. Other candidates
fall in the second category that mentioned, as treatments for diseases such as
cancer and AIDS that work by boosting the patient's internal defense systems.
For now, the selection of disease targets is limited by the available science
and technology. As we learn more and more about regulation of gene expression
in the normal organism, we will be able to apply this to our understanding of
disease processes. Today’s biomedical investigator has, for the first time, the
scientific knowledge and technological tools to begin addressing questions that
have eluded us in the past.
CONCLUSION
Gene therapy is a novel
method of treating some of the hitherto untreatable diseases. It involves the
introduction of a functional gene to biologically active proteins can be
synthesized with in the cells whose function is to be altered, introduced as a
concept about two decades ago it has become a reality today .
A verity of DNA delivery
systems have been developed involving biological, physical and chemical agents.
Gene therapy was initially through to be a treatment modality for inherited
single gene defects however it has also found applications in acquired
diseases.
Its use is being studies
in the treatment of cancer, immunodeficiency diseases, cardiovascular,
metabolic, neurological disorders; hormones and blood factors deficiencies. It
is also being developed as a “gene” vaccine against influenza and malaria.
Recently attempts have been made for its use in treatment of HIV infection.
Gene therapy, although
still in the infant stages of development offers the possibility for major
advances in prevention and treatment of these diseases. Presently the clinical
application of gene therapy is limited by the availability of suitable gene
transfer methodology:
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LIST OF ABBREVIATIONS
AA=Amino-acid
Cdk= Cyclin-dependent kinase
ER= Endoplasmic reticulum
ES cells = Embryonic stem cells
GH= Growth hormone
GK rat= Goto-Kakizaki rat
GLUT= Glucose transporter
HGF= Hepatocyte growth factor
HIV= Human immunodeficiency virus
HNF= Hepatocyte nuclear factor
HSV= Herpes simplex virus
IGF= Insulin-like growth factor
IL= Interleukin
IRR=Insulin
receptor-related receptor
IRS= Insulin receptor substrate
LIF= Leukemia inhibitory factor
NOD= Nonobese
Pdx= Pancreatic and duodenal homeobox gene
PERV=Pig endogenous
retroviruses
PRL= Prolactin
Reg= Regenerating protein
STZ= Streptozotocin
TGF= Transforming growth factor
VEGF=Vascular
endothelial growth factor
VSV-G=Vesicular
stomatitis virus G glycoprotein.
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