Applications of Gene Therapy

8/12/2019 Applications of Gene Therapy

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!"#$%& %()% *)+)"# *,! Certain wavelengths of radiation

ionizing radiation such as gamma rays and X-rays

ultraviolet rays, especially the UV-C rays (~260 nm) that areabsorbed strongly by DNA but also the longer-wavelength UV-Bthat penetrates the ozone shield.

Highly-reactive oxygen radicalsproduced during normalcellular respiration as well as by other biochemical pathways.

Chemicals in the environment many hydrocarbons, including some found in cigarette smoke

some plant and microbial products, e.g. the aflatoxins producedin moldy peanuts

Chemicals used in chemotherapy, especially chemotherapyof cancers


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All four of the bases in DNA (A, T, C, G) can be

covalently modified at various positions.

One of the most frequent is the loss of an amino

group ("deamination") resulting, for example, in a Cbeing converted to a U.

Mismatchesof the normal bases because of a failure ofproofreading during DNA replication.

Common example: incorporation of the pyrimidine U(normally found only in RNA) instead of T.


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Breaksin the backbone.

Can be limited to one of the two strands (a single-

stranded break, SSB) or

on both strands(a double-stranded break (DSB).

Ionizing radiation is a frequent cause, but some

chemicals produce breaks as well.

CrosslinksCovalent linkages can be formed betweenbases

on the same DNA strand ("intrastrand") or

on the opposite strand ("interstrand"). Several chemotherapeutic drugs used against cancers

crosslink DNA .


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NER differs from BER in several ways.

It uses different enzymes.

Even though there may be only a single "bad"

base to correct, its nucleotide is removed alongwith many other adjacent nucleotides; that is, NERremoves a large "patch" around the damage.

The damage is recognized by one or more proteinfactors that assemble at the location.

The DNA is unwound producing a "bubble". Theenzyme system that does this is TranscriptionFactor IIH, TFIIH, (which also functions in normaltranscription).

Cuts are made on both the 3' side and the 5' sideof the damaged area so the tract containing thedamage can be removed.

A fresh burst of DNA synthesis using the intact(opposite) strand as a template fills in thecorrect nucleotides. The DNA polymerasesresponsible are designated polymerase deltaandepsilon.

A DNA ligasecovalently inserts the fresh pieceinto the backbone.


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Normal stem cells are characterized by three

properties:

1 Capability of self-renewal2 Strict control on stem cell numbers

3 Ability to divide and differentiate to generate all

functional elements of that particular tissue

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Hierarchy of stem cells with cell determination,

differentiation and maturation.It also shows potential areas of

A.Trans-germal plasticitydifferentiation from one

stem to other stem cell type;

B.De-differentiationregression of a fixed lineagecell type to a more primitive cell type;

C.Trans-determinationdifferentiation from one

progenitor cells to another; and

D.Trans-differentiationhypothetical differentiation

of one cell type to another without

dedifferentiation.


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POTENTIAL TREATMENTS

Brain damage:--Parkinsons disease andAlzheimer's

Various cancers

Spinal cord injury

Heart damage

Blindness and vision impairment

ALS amyotrophic lateral sclerosis (ALS)

Orthopaedic Infertility

Wound healing


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The most common and successful cell

based therapy is Bone marrow transplant

Dates back to the work of Mc Culloch and

Till, Canadian scientists in 1961:

Bone marrow from one inbred mouse

another mouse whose bone marrow isdestroyed


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To restore damaged HSC in radiotherapy

or chemotherapy for cancer

Treat diseases due to defective

hematopoietic tissue-

Luekaemia

AnemiaThalassaemia


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Source..-HSCs are found in the bone marrow of adults, which

includes femurs, hip, ribs, sternum, and other bones.

-Cells can be obtained directly by removal from the hip

using a needle and syringe, or

-from the blood following pre-treatment with cytokines, suchas G-CSF (granulocyte colony-stimulating factors), that

induce cells to be released from the bone marrow

compartment
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The hematopoietic tissue contains cells with

long-term and short-term regeneration

capacities and committed multipotent,

oligopotent, and unipotent progenitors.


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a small number of HSCs can expand to generate a

very large number of progeny HSCs. This

phenomenon is used in bone marrow transplant

when a small number of HSCs reconstitute the

hematopoietic system.

Stem cell self-renewal is thought to occur in the stem

cell niche in the bone marrow

What makes HSC a better candidate for cell therapy?


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O(.F#"1-"1 '#1+(1+Parkinson's diseaseis a degenerative disorder of the central

nervous system that often impairs the sufferer's motor skills,

speech, and other functions

Parkinson's disease belongs to a group of conditions called movement

disorders.

It is characterized by muscle rigidity, tremor, postural abnormalities, gait

abnormalities, a slowing of physical movement (bradykinesia) and a loss ofphysical movement (akinesia) in extreme cases.

The primary symptoms are the results of decreased stimulation of the

motor cortex by the basal ganglia.

Normally this involves insufficient formation and thus action of dopamine

produced in the dopaminergic neurons of the midbrain (specifically the

substantia nigra).

Secondary symptoms may include high level cognitive dysfunction andsubtle language problems. PD is both chronic and progressive.


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Neuronal Pathways that Degenerate in Parkinson's Disease.Signals that control body movements travel along neurons that

project from the substantia nigra to the caudate nucleus and

putamen (collectively called the striatum). These "nigro-striatal"neurons release dopamine at their targets in the striatum. InParkinson's patients, dopamine neurons in the nigro-striatal pathway

degenerate for unknown reasons.


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Present treatment for parkinsons:

Most patients suffering from Parkinson's Disease are treated

with a drug called levodopa, which the brain converts intodopamine.

It initially helps most patients, but unfortunately, side effects

of the drug increase over time and its effectiveness wanes.


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U$+) cell based therapy for Parkinson disease

Therapy uses two fundamental strategies

1. Cell Implantation strategy=Use of undifferentiated stem cells to

differentiate into the cell type of interest

neuronal precursor cells- grow them in vitro into desired cell type

and implant to body

Use of pluripotent embryonic stem cells and exploiting its ability to

form any cells.

2. Trophic factor strategy

The other repair strategy relies on finding growth hormones andother "trophic factors"(signaling molecules ) that help stem cell

to survive and grow

The signalling molecules can fire up a patient's own stem cells and

endogenous repair mechanisms


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Surgical approach :

Transplanting dopamine-producing chromaffin cells

from patients' own adrenal glands to the nigro-striatalarea of their brains.

Problems outweigh the benefits:

very modest and inconsistent improvement in their

patients' symptoms,

Gains disappeared within a year after surgery

Risk associated with the procedurewhich required

both brain and abdominal surgery on patients who are

often frail and elderly.


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Stem cell based approaches

Fetal Tissue Transplants in Parkinson's Disease Research

Strategy: Implant cells into the brain that can replace the

lost dopamine-releasing neurons

Problem:

Fully developed and differentiated dopamine neurons do

not survive transplantation- so differentiated tissue

transplant is not possible.

Moreover, full functional recovery depends on the

appropriate connections with the normal target neurons inthe striatum.


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Fetal tissue transplant

The strategy based on transplanting developing

dopamine neurons from fetal brain tissue, has fared

better

fetal tissue transplanted directly from the developing

nigro-striatal pathways of embryonic mice into the

anterior chamber of an adult rat's eye continues to

mature into fully developed dopamine neurons.

Reduced parkinsons like symptoms


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Raising Neurons for Replacement in Patients withParkinson's Disease

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"Cancer Stem Cells.

Why a tumor does not respond to treatment?

Why tumors recur?

Why cancer cells develop resistance to treatment?

At present, the shrinkage in the size of a tumor isconsidered as a response to the treatment. However,tumor often shrinks in response to the treatment only to

recur again.

These and many other raised questions may beanswered by the new concept of "Cancer Stem Cells.


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Living connective tissue made up of cellssuspended in a matrix

Bones are also called osseous tissue, (Latin:"os")

Adults have 206 bones. At birth there areabout 270, the number decrease as bonesfuse.

The skeletal system is divide into 2 regions: Axial skeletoncentral axis

Appendicular skeletonthe limbs(extremities)

Possesses cells, nerves, blood vessels andpain receptors


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Long bones

Central, usually hollow tubular region linkedto epiphysis by metaphysis.

Cavity filled with bone marrow

Shaft walls made of hard compact bone,

thickest in the middle.

Short bones

Found in the wrist and ankle, carpals and

tarsals.

No shaft; do not increase dramatically in size

during growth, cubical in shape.

Cancellous bone in the center; hard outer shell

of compact bone.


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Bone Cells

The osteoblast

large cells (20-30 m), in the form of a polyhedron, with a

basophilic cytoplasm, and with a substantial rough

endoplasmic reticulum and Golgi apparatus

communicate with the osteocyte network and neighbouring

osteoblasts

synthesize the organic matrix or osteoid material and

express ALP

the intercellular connection is Connexin 43

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The osteocyte

some osteoblasts remain trapped within the matrix after

mineralisation, becoming transformed into osteocytes

on the interior surface of bone and the most abundant cells in bone

stellate and are found in the interior of lacunae

the cytoplasmic processes communicate with each other throughbone canaliculi filled with extracellular bone fluid

the osteocytes organize themselves into a syncytium of

interconnected cells

When trauma occurs in the bone, the cessation in the blood supply

causes hypoxia and necrosis of the osteocytes

control bone remodeling, detecting the mechanical variations of the

loads, a phenomena known as mechanotransduction


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The osteoclast

the cells responsible for resorption

large cells (100 m), multinucleate, rich in mitochondria and vacuoles

Contain TRAP, which permits the dephosphorylation of the proteins

possess receptors for calcitonin

originate from the bone marrow hematopoietic stem cells known as

Granulocyte-Macrophage Colony-Forming Units (GM-CFU), precursors of

macrophages and monocytes

two special features in the membrane: a ruffled border and a clear area rich

in microfilaments, with integrins .


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Pagets Disease:

Caused by increased remodelling of the bone.

Results in bone with poor architectural quality. Wild arrangement of trabeculae

Disorganized lamallae, primitive woven bone in cortical region.

Confined to single bone in the bodynot systemic.

Results in pain, loss of function and fracture.

Osteosarcoma:

Bone gradually replaced by tumour cells, which may be partially calcified but

generally disorganized.

Effects on bone properties: Areas affeced by the tumour will have greatly reduced mechanical properties.

Tumours can completely penetrate the cortical region of the affected bone and

invade the soft tissue with only a thinperiosteal shell separating the tumour and

the surrounding tissue.

Fractures occur with minimal and at times zero trauma at the tumour site.


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Fracture Risk:

Depends on

Inherent strength of load Geometry

Material properties

Applied load:

Direction, rate and model of loading. Magnitude of load.

Fracture risk increases with age due to age and hormonal related changes in

bone properties.

Increased porosity and decreased geometric properties.

Decreased fatigue resistance.

Fracture risk also increases with age due to more frequent adverse loading

events (falls) and decreased energy adsorption.

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