Gene therapy for Parkinson’s disease

Under the esteemed guidance of:

Mr. Bimalendu Chowdhury

Astt.Professor

Department of pharmacology

RIPS

Presented by: Utkarsh Alok

M.Pharm, 1st sem Department of pharmacology

RIPS

INTRODUCTION ON PARKINSON’S DISEASE:

Parkinson’s disease is a progressive neurodegenerative disorder whose main histo-pathological feature is the loss of dopaminergic neurons in the pars compacta of the substantia nigra with secondary striatal dopaminergic insufficiency[1].

Parkinsonism is a clinical syndrome consisting of four cardinal feature:1. Bradykinesia (slowness and poverty of movement),2. Muscular rigidity, 3. Resting tremor (which usually abates during voluntary

movement), 4. Impairment of postural balance leading to disturbances of

gait and falling.[2]

Cognitive and psychiatric dysfunctions like dementia anddepression also accompanies the clinical symptoms.[8]

WHAT CAUSES PARKINSON'S DISEASE?

•Progressive loss of brain cells (neurones) in a part of the brain called the substantia nigra, which produces the chemical dopamine.•As the cells die, less dopamine is produced and transported to the striatum, the area of the brain that co-ordinates movement. •Symptoms develop as neurones die off and dopamine levels drop.

ETIOLOGIC FACTORS

•Aging: The annual incidence of IPD increases with age from about 20 per 100,000 persons in the fifth decade of life to about 90 per 100,000 persons in the seventh decade of life, with the usual age of onset at about age 60[17].•Environmental factor: rural living, drinking well water, and heavy metal and hydrocarbon exposure have small but demonstrable contributions . Exposure to MPTP is capable of inducing Parkinsonism in humans [3].•Genetic factors: several gene mutations that can cause the disease directly. Some of these mutations involve genes that play a role in dopamine cell functions. Parkinson’s has developed at an early age in individuals with mutations in genes for alpha-synuclein, Parkin, PINK1, DJ-1 etc.

PATHOLOGY

1.NEURON LOSS- even mildly affected PD patients have lost about 60% of their DA neurons, and it is this loss, in addition to possible dysfunction of the remaining neurons, that accounts for the approximately 80% loss of DA in the corpus striatum.[3]

2.LEWY BODIES- major antigenic feature of Lewy bodies is the expression of cellular proteins involved in protein degradation. Presence of these antigens has been hypothesized to represent efforts on the part of the cell to degrade the abnormal protein aggregate [3].

PATHOGENETIC MECHANISM

Free radical and deficits in energy metabolism:This theory is also referred to as the oxidant stress hypothesis or the endogenous toxin hypothesis- initiated by H2O2, O2, which are produced as a byproduct of mitochondrial oxidative phosphorylation.Protective mechanism- superoxide dimutase (and catalase, antioxidants like ascorbic acid, tocopherol etc.Mutation in SOD cause accumulation of aggregates of misfolded mutated SOD, this can cause degeneration of motor neurons.

•The pharmacological and surgical therapies described above aim to improve the symptoms of PD but none are proven to have a significant impact on the underlying disease process with respect to either slowing disease progression or restoring the affected dopaminergic neurons.• Gene therapy has distinct potential advantages over conventional treatment modalities for PD as it could theoretically be used to preserve or restore dopaminergic neurons affected by PD through the action of neurotrophic factors[41] or alternatively increase the availability of enzymes required for dopamine synthesis[43]. •Although the disease modifying properties of these therapies remains to be proven, they could potentially target the underlying patho-physiological imbalances and may result in much less fluctuation in response and a lower prevalence of dyskinesias than conventional pharmacotherapy for PD.

Why gene therapy?

What Are Genes?

• Genes are carried on chromosomes and are the basic physical and functional units of heredity .

• Genes are specific sequences of bases that encode instructions on how to make proteins.

• When genes are altered so that the encoded proteins are unable to carry out their normal functions, genetic disorders result.

Gene therapy is a new approach to treating medical conditions, which can be described as the use of genes as drugs.Gene therapy can also be used to treat disorders where the genetic cause is not known, or may not be caused exclusively by genetic defects, such as Parkinson’s[44]. The carrier particle or molecule used to deliver genes are called as vectors.

DEFINATION: Gene therapy is the process of inserting genes into cell to treat diseases, where newly introduced genes will encode proteins and correct the deficiencies[45].

What Is Gene Therapy?

What Is Gene Therapy?

•In most gene therapy studies, a "normal" gene is inserted into the genome to replace an "abnormal," disease-causing gene. •A carrier molecule called a vector must be used to deliver the therapeutic gene to the patient's target cells. •The most common vector is a virus that has been genetically altered to carry normal human DNA. •Viruses have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner. •Scientists manipulate the virus genome to remove disease-causing genes and insert therapeutic ones. •Target cells, such as the patient's liver or lung cells, are infected with the viral vector.

How Does Gene Therapy Work?

How Does Gene Therapy Work?

Approaches for gene therapy

There are two approaches to achieve gene therapy:-

1. Somatic cell gene therapy.2. Germ cell gene therapy.

Somatic cell gene therapy: The therapeutic genes are transferred into the somatic cell or body, of a patient. Any modifications and effects will be restricted to the individual patient only, and will not be inherited by the patient's offspring or later generations. Somatic gene therapy represents the mainstream line of current basic and clinical research, where DNA is used to treat a disease in an individual[47].

Germ cell gene therapy.

Germ cells, i.e. sperm or eggs, are modified by the introduction of functional genes, which are integrated into their genomes. This would allow the therapy to be heritable and passed on to later generations. Although this should, in theory, be highly effective in counteracting genetic disorders and hereditary diseases.

The genetic alteration in somatic cell is not carried to the next generation. Therefore , somatic cell gene therapy is preferred and extensively studied with an ultimate objective of correcting human disease [45].

• Problems With Gene Therapy.

•Short-lived nature of gene therapy- patients will have to undergo multiple rounds of gene therapy.

•Immune response- risk of stimulating the immune system in a way that reduces gene therapy effectiveness is always a potential risk.

•Problems with viral vectors- viruses, the carrier of choice, present potential problems to the patient, like toxicity, immune and inflammatory responses, and gene control and targeting.

•Multi-gene disorders- most common disorders, such as heart disease, high blood pressure, Alzheimer's disease, arthritis and diabetes, are caused by the combined effects of variations in many genes.

•Chance of inducing a tumor (insertional mutagenesis) - If the DNA is integrated in the wrong place in the genome, for example in a tumor suppressor gene , it could induce a tumor.

As such, gene therapy involves a great risk. There are several regulatory agencies whose permission must be sought before undertaking any work related to gene therapy. Recombinant DNA Advisory Committee (RAC) is the supervisory body of the National Institute of Health (NIH), USA, that clears proposals on experiments involving gene therapy[45].

TYPES OF GENE THERAPY

1.Ex.vivo gene therapy.2.In-vivo gene therapy.

Ex.vivo gene therapy: Cells are modified outside the body and then transplanted back in again. Ex vivo gene therapy uses gene transfer techniques to produce genetically modified primary cells or cell lines for transplantation into the brain of Parkinsonian individuals.This procedure involves patients own cell for correction and then their return back to the patient. This technique is therefore, not associated with adverse immunological responses after transplanting cells. Ex-vivo gene therapy is efficient only, if the therapeutic gene is stably incorporated and continuously expressed, this can be achieved by use of vectors.

In vivo gene therapy: In vivo gene therapy for the CNS consists of the direct intra-cerebral injection of genetic material using appropriate vectors[54].The success of in-vivo gene therapy depends upon the following parameters[45]:-

•The efficiency of the uptake of the remedial (therapeutic gene) by the target cells.

•The expression capability of the gene.

Gene transfer to the brain by using viral vectors offers the advantage of being less invasive than transplantation techniques, leaving the striatal circuitry undisturbed by cellular implants and eliminating risks of unwelcome host immune responses or tumour formation[53].

Vectors for gene delivery

The carrier particle or molecule used to deliver genes are called as vectors.

The ideal vector system would have the following characteristics:

an adequate carrying capacity;

to be undetectable by the immune system;

to be non-inflammatory;

to be safe to the patients with pre-existing lung inflammation to have an efficiency sufficient to correct the cystic fibrosis phenotype

to have long duration of expression and/or the ability to be safely re-administered.

There are two categories of delivery vehicle (‘vector’)

1.Non-viral vectors2.Viral vectors

Retrovirus Herpes Sim-plex V

Adenovirus AAV Lipo-some

DNA Polymer

Integration Yes Non Non Yes Non

Expression Stable Transient Transient Stable Transient

Transfection Efficient Efficient Efficient Low Low

Immune Response No Yes High No Yes Yes or No No

Generally, viral vector system show higher gene transfer efficiency than non-viral gene carrier system, but viral systems have potential risk of wild type virus regeneration, immunogenecity and cancer formation.

Vectors associated with gene therapy for parkinsons:-

Overexpression of GDNF mediated by the lentiviral vector has conferred some protection of the nigrostriatal dopamine terminals against toxic insults[63].

Ad-GDNF can protect dopaminergic neurons and improve dopamine-dependent behavioral function in young rats with progressive 6-OHDA lesions of the nigrostriatal projection[71].

AAV-2, when administered locally, it transduces only neurons within the central nervous system and is particularly efficient in brain regions known to be involved in the pathophysiology of PD, such as the globus pallidus and substantia nigra[75].

Strategies for the rationality in the gene therapy trials in Parkinson’s disease

PD has a complex pathophysiology that is by no means fully

understood and involves multiple brain structures and signalling

pathways. There are three broad approaches to selection of a

therapeutic target[81].

1. Restoration of DA synthesis in the dorsal striatum;

2. Modulation of activity in the basal ganglia downstream of the

striatum; and

3. Modification of disease progression by neuroprotection.