Gene Therapy: Principles and Types

Gene therapy uses genes or short oligonucleotide sequences as therapeutic molecules, instead of conventional drug compounds.
It treats defective genes contributing to disease development.
It involves introducing foreign genes into an organism to treat hereditary or acquired genetic defects.
Treats disease with minimal toxicity, by the expression of the inserted DNA by the cell machinery.
The first approved gene therapy procedure was performed on Sept 14, 1990 by W. French Anderson and his colleague at NIH.
They treated Ashanthi de Silva, a four-year-old girl born with SCID (severe combined immune deficiency) due to a lack of the enzyme adenosine deaminase (ADA).
Gene therapy replaces defective genes with functional ones so the body can make functional proteins and eliminate the root cause of disease.
The aims are to replace faulty/defective genes, silence faulty genes or introduce new normal genes

Approaches include inserting a normal gene into a specific location within the genome to replace a non-functional gene.
Gene therapy is classified into two types: somatic and germ line.

Somatic cells of a patient are targeted for foreign gene transfer.
Effects caused by the foreign gene are restricted to the individual patient only and not inherited by offspring.

Functional genes are integrated into the genomes and inserted into germ cells (sperm or eggs).
Targeting germ cells makes the therapy heritable.

In GAT, the simple addition of functional alleles is used to treat inherited disorders caused by the genetic deficiency of a gene product.
GAT has been applied to autosomal recessive disorders.
Dominantly inherited disorders are much less amenable to GAT.

Genes encoding toxic compounds (suicide genes) or prodrugs (reagents which confer sensitivity to subsequent treatment with a drug) are utilized to kill the transfected/transformed cells.
This is popular in cancer gene therapies.

Blocks the expression of any diseased gene or a new gene expressing a protein harmful to a cell.
Suitable for treating infectious diseases and some cancers.

Corrects a defective gene to restore its function at a genetic level by homologous recombination, or at an mRNA level using therapeutic ribozymes or therapeutic RNA editing.

There are two approaches for the transfer of genes in gene therapy: transfer of genes into patient cells outside the body (ex vivo gene therapy) and transfer of genes directly to cells inside the body (in vivo gene therapy).

Genes are transferred to cells grown in culture, transformed cells are selected, multiplied, and then introduced into the patient.
The use of autologous cells avoids immune system rejection of the introduced cells.
Cells are sourced initially from the patient, grown in culture, and reintroduced into the same individual.
Can be applied to tissues like hematopoietic cells and skin cells, which can be removed, genetically corrected outside the body, and reintroduced into the patient body, where they become engrafted and survive for a long time.

Cloned genes are transferred directly into the tissues of the patient.
This is done in the case of tissues whose cells cannot be cultured in vitro in sufficient numbers (like brain cells) or where reimplantation of the cultured cells is not efficient.
Liposomes and certain viral vectors are employed for this purpose due to of a lack of any other mode of selection.
Cultured cells infected with recombinant retrovirus in vitro to produce modified viral vectors regularly are often used.
These cultured cells transferred the gene to surrounding disease cells
The efficiency of gene transfer and expression determines the success, because of the lack of any way for selection or amplification of cells which take up and express the foreign gene.

In vivo is less invasive, and technically simple with vectors introduced directly, where no safety check is possible and with no control but decreased control is shown
Ex vivo is more invasive and technically more complexity with no vectors being introduced and a closer control but there could be increased problems

In cancer, the target cells are tumor cells, antigen presenting cells (APCs), blood progenitor cells, T cells, fibroblasts, muscle cells.
For inherited monogenic disease, rheumatoid arthritis, or infectious disease the target is T cells, blood progenitor cells, antigen presenting cells (APCs), muscle cells
For Cardiovascular disease, the target is Endothelial cells, muscle cells

A carrier molecule, called a vector, must deliver the therapeutic gene to the patient's target cells.

Viruses bind to their hosts and introduce their genetic material into the host cell.
A viral vector is a virus modified in a laboratory environment to introduce genetic material into a cell; viral genes that cause diease are removed and replaced with genes encoding effect
Viral vectors are target specific, good at delivering genetic material, some integrate into the host genome, and can be modified.
Gene gun employs a high-pressure delivery system to shoot tissue with gold or tungsten particles coated with DNA.
Microinjection uses a glass micropipette to insert microscopic substances into a single living cell.
Performed under a specialized optical microscope setup called a micromanipulator.

Recombinant retroviruses can integrate into the host genome in a stable fashion.
Can carry a DNA size of less than 3.4kb.
They are replication defective virus particles and targets a dividing cell

A human virus capable of integrating into chromosome 19.
Single-stranded, non-pathogenic small DNA virus.
Enters the host cell, becomes double-stranded, and gets integrated into the chromosome.

Are lipid DNA complexes where the DNA construct is surrounded by an artificial lipid layer.
Most of it gets degraded by lysosomes.

A synthetic conjugate using poly-L-lysine bound to a specific target cell receptor.
Therapeutic DNA is made to combine with the conjugate to form a complex.
Avoids lysosomal breakdown of DNA.