Gene Therapy

Gene therapy

Any procedure intended to treat or alleviate diseases by genetically modifying the cells of a patient

In vivo

Genetic material transferred directly into patient’s cells

Ex vivo/in vitro

Cells removed from a patient, genetic material inserted, modified cells placed back into patient

What are 4 major diseases?

1. Infectious disease
2. Cancer
3. Inherited disorders
4. Immune system disorders

Infectious diseases

Infection by a virus or bacterial pathogen

Cancers

\-Genetic disease

\-Continuous cell division resulting in activation of a oncogene or inactivation of a tumor suppressor gene

Inherited disorders

Genetic deficiency

Immune system disorders

\-Allergies, inflammations, autoimmune diseases

\-Body cells are inappropriately destroyed by immune system cells

What are the 4 kinds of gene therapy strategies?

1. Gene augmentation therapy (GAT)
2. Targeted killing of specific cells
3. Targeted mutation correction
4. Targeted inhibition of gene expression

When is gene augmentation therapy used?

Diseases caused by loss of function of a gene

Gene augmentation therapy (GAT)

Giving cells a wild type copy to restore normal phenotype

When is targeted killing of specific cells used?

Cancer gene therapies

Targeted killing of specific cells

Genes are directed to target cells then expressed to kill the cell

What are 4 ways for targeted killing of specific cells?

1. Suicide gene
2. Prodrug
3. Foreign antigen gene
4. Cytokine gene

Suicide gene

Inserted genes are expressed to produce a lethal toxin

Prodrug

Gene makes cells susceptible to a drug that will kill them

Foreign antigen gene

Stimulates immune response

Cytokine gene

Amps up immune system in either diseased or nondiseased cells (especially immune system cells)

When is targeted mutation correction used?

Inherited mutation produces a dominant negative effect

What two levels can targeted mutation correction be performed?

1. At the gene level
2. At the RNA transcript level

When is targeted inhibition of gene expression used?

Dominant mutation or overexpression of a gene

Targeted inhibition of gene expression

Blocks expression of a single gene at DNA, RNA, or protein levels

Why is current gene therapy only for somatic mutations and not germline mutations?

\-Currently unethical

\-No consent from child since all cells would be affected

Somatic mutations

\-Can occur in nongermline tissues

\-Can’t be inherited

Germline mutation

\-Present in egg or sperm

\-Can be inherited

Ex vivo gene therapy

\-Cells modified outside the body then implanted back into the patient

\-Uses autologous (patient) or allogenic (not patient) cells

In vivo gene transfer

\-Cloned genes are transferred directly into the tissues of patient

\-Useful when individual cells can’t be cultured in vitro (ex. brain cells)

Episomes

\-Inserted genes that remain extrachromosomal

\-Not guaranteed that both daughter cells will have the gene

What are the advantages of genes integrated into chromosomes?

\-Gene will be passed onto daughter cells

\-Long-term, stable expression

\-Possibility of a cure for some disorders

What are the disadvantages of genes integrated into chromosomes?

\-Insertion occurs almost randomly so location can vary

\-Inserted genes may not be expressed

\-Inserted genes could cause death of the host cell

\-Possibility of cancer

What are the advantages of nonintegrated (episomal) genes

Good for short-term treatment (ex. cancer)

What are the disadvantages of nonintegrated (episomal) genes

\-Gene may not be passed onto both daughter cells

\-Repeated treatments needed

What are the two main types of vectors?

1. Viral
2. Nonviral

Oncoretroviral vectors

\-Retrovirus (RNA → DNA)

\-Very efficient at transferring DNA into cells

\-Used with cancer therapy

Adenovirus vectors

\-Produce infections of upper respiratory and GI tract

\-Evolved spike protein that causes cell to unknowingly bring in virus

\-Large virus (large inserts)

\-Can infect a variety of cell types

Adenovirus disadvantages

\-Short lived

\-Non-target cells at risk

\-Risk of immune response

Adenoassociated virus vectors (AAVs)

\-Small, single-stranded DNA viruses that require a helper virus in order to infect

\-Only contain gene of interest

Herpes vectors (HSV)

\-Affinity for CNS

\-Large insert capability

\-Non-integrating so no long term expression

\-Used for delivering genes into neurons

Lentiviruses

\-Retrovirus that infect macrophages and lymphocytes

\-Integrate directly into chromosome

\-Transduce nondividing cells

Liposomes

\-Fat molecules

\-Can get DNA into cells by fusing membrane

Liposome advantages

\-DNA/lipid complexes easy to prepare

\-No limit to DNA size

Liposome disadvantages

\-Low efficiency

\-DNA not designed to integrate into chromosomal DNA

Direct injection/particle bombardment

\-DNA injected directly with a syringe and needle or with a gene gun

\-Good for in vivo cells (like brain cells)

Gene gun (particle bombardment)

DNA coated onto metal pellets and fired directly into tissues

Advantages of direct injection/particle bombardment

Simple and safe

Disadvantages of direct injection/particle bombardment

\-Poor efficiency of gene transfer

\-Low level of stable integration

Receptor-mediated endocytosis

\-DNA is coupled with a targeting molecule and brought in through endocytosis

\-Uses transferrin receptor

Advantages of receptor-mediated endocytosis

\-High gene transfer efficiency

\-Good for multiple cell types

Disadvantages of receptor-mediated endocytosis

\-No integration of transferred genes

\-Not completely stable

\-DNA may be degraded

Single gene disorders

Individuals are severely affected and no effective treatment

Recessively inherited disordered

\-Simple deficiency of a specific gene product

\-Usually loss-of-function mutations

ADA deficiency (adenosine deaminase)

\-first gene therapy trial for an inherited disorder (1990)

\-results in immunodeficiency (T cells)

ADA gene therapy steps

1. Cloning a normal gene into retroviral vector
2. Transfecting ADA recombinant into cultured ADA T cells from patient
3. Identify resulting ADA+ T cells and expand
4. Reimplant cells back into patient

Cancer gene therapies

\-Many based on targeted killing of diseased cells

\-Target single genes (like tumor suppressor genes)

\-Prodrug genes

Gene therapy for infectious disorders

\-Provoke a specific immune response

\-Target life-cycle of infectious agent

What are three strategies to interfere with the life cycle of an infectious agent?

1. Blocking HIV-1 infection
2. Inhibition at RNA level
3. Inhibition at protein level

Blocking HIV-1 infection

\-HIV-1 normally infects T cells by binding viral gene to cell receptor

\-Transfer of a soluble form of receptor antigen into T cells causes circulating receptors

\-HIV binds to circulating receptors instead of T cells

Inhibition at the RNA level

Antisense genes created so viral RNA unable to be expressed

Inhibition at protein level

\-Genes introduced that code for dominant-negative mutant HIV proteins

\-Bind and inactivate HIV proteins