GENE THERAPY


GENE THERAPY

The use of recombinant genetic information (DNA, RNA, hybrid molecules) under different forms of pharmaceutical preparations as therapeutic agent.

Can offer promise for treating currently intractable diseases.

Can be used for both inherited monogenic disorders and acquired diseases.

Take a gene from a patient - EX VIVO.

Isolate the target cells.

Transfer the gene to them.

Reinfuse the gene-modified cells into the patient.

Infuse the therapeutic gene inside a vector into the patient - IN VIVO.

TARGET TISSUES

Haematopoietic system: stem cells, progenitor cells, mature cells

Muscle: myoblasts, myofibres

Liver: hepatocytes

Vasculature: endothelium, smooth muscle

Skin: keratinocytes, fibroblasts

CNS: neurons, glia, microglia

Lung: epithelium

IDEAL TARGET CELL: pluripotent, self-regenerating stem cells from the patient

VIRAL GENE THERAPY VECTORS

ADENOVIRUS: non-integrating

ADENO-ASSOCIATED VIRUS: non-integrating

RETROVIRUS: integrating

LENTIVIRUS: integrating

HUMAN GENE THERAPY SUCESSES VIA EX VIVO PROCEDURE:

Severe combined immune deficiencies SCID - haematopoietic stem cells X-linked chronic granulomatous disease CGD - haematopoietic stem cells

Junctinoal epidermiolysis bulls JEB - epidermal stem cell

X-linked adrenoleukodystrophy ALD - haematopoietic stem cells

Metachromatic leukodystrophy MLD - haematopoietic stem cells

B-thalassemia/sicke cell disease SCD - haematopoietic stem cells

HUMAN GENE THERAPY SUCESSES VIA IN VIVO PROCEDURE

Haemophilia B - AAV vector

Parkinson’s disease - AAV vector

Leber’s congenital amaurosis - AAV vector

EX VIVO PROCEDURES:

X-SCID

X-linked severe combined immunodeficiency

Complete lack of NK and T-cells in the peripheral blood.

Mutation of gamma chain LR2RG a subunit of haematopoietic cytokine receptors IL2, IL4, IL7, IL9, IL15, IL21: result, block in T-cell development.

Children with XSCID usually die in their first month of life

EX VIVO gene devilry protocol

  1. bone marrow harvested, CD34 positive stem cells are isolated

  2. cells activated to grow for 24h with cytokines: need to activate mitosis as retroviruses will only transduce dividing cells

  3. cells traduced by three rounds of culture int he presence of the supernatant containing IL2RG retroviral vector in sterile bags

  4. transducer CD34+ cells infused back into the patient

WISKOTT - ALDRICH SYNDROME WAS

Infections, microthrombocytopenia, eczema, autoimmunity lymphoid malignancies.

X-linked, mutations in the WAS gene, codes for WASP a protein that regulates the cytoskeleton.

WASP-defective immune cells, alterations in proliferative responses after activation, cell migratino, immunological synapsis formation, cytotoxicity.

Regular treatment is allogenic haematopoietic stem cell transplantation.

B-HAEMOGLOBINOPATHIES

Require the introduction of a normal functioning B-globin transcription unit in to the haematopoietic stem cells of the patient. A minimum of 25% of normal levels of B-globin are required for the therapeutic effect; 50% are curative.

Lentiviral vectors.

Ex vivo protocol.

No current lentiviral vector capable of curing thalassemia major.

IN VIVO PROCEDURES:

LEBER’S CONGENITAL AMAUROSIS

In vivo delivery to the retina of AAV vector with RPE65 gene

HAEMOPHILIA B

X linked bleeding disorder, the royal disease, defect in coagulation factor IX.

Administratin of a AAV vector into peripheral veins targeting liver.

Results in transgene expression sufficient to improve bleeding phenotype over at least 3 years.

#genomics