1. Gene Therapy of Hematopoietic Disorders
Prepared By/ Hussein M. Dakka
Supervisor / Ahmad Sh. Silmi

2. Part I: Gene Therapy
Somatic cell gene transfer may be used to correct a deficit in a cell, to enhance or inhibit its pre-existing function, or to provide the cell with an entirely new capacity.

3. Essential Requirements of Gene therapy for an Inherited Disorder
Identity of the molecular defect
A functional copy of the gene
An appropriate vector
Knowledge of the pathophysiological mechanism
Favorable risk-to-benefit ratio
An appropriate target cell
Strong evidence of efficacy and safety
Regulatory approval

4. Gene Transfer Strategies

5. Isolation of Pure Hematopoietic Stem Cells

6. The Target Cell
The ideal target cells are stem cells or progenitor cells taken from the patient.
Introduction of the gene into stem cells can result in the expression of the transferred gene in a large population of daughter cells.
An important logistical consideration is the number of cells into which the gene must be introduced in order to have a significant therapeutic effect.
To treat PKU, for example, the approximate number of liver cells into which the phenylalanine hydroxylase gene would have to be transferred is approximately 5% of the hepatocyte mass, or approximately 10^10 cells,
although this number could be much less if the level of expression of the transferred gene is higher than wild type.

7. DNA Transfer into Cells: Viral Vectors
The ideal vector for gene therapy would be safe, readily made, and easily introduced into the appropriate target tissue, and it would express the gene of interest for life.
None of the vectors currently available is suitable for everynpotential application of gene transfer in haematological disease, and none is free of significant limitations and adverse effects.
Ultimately, therefore, entirely new synthetic or semisynthetic vectors will have to be developed. Possibilities include the generation of hybrid viral vectors.
Here, I briefly review three of the most widely used classes of viral vectors, those derived from
Retroviruses, Adeno-associated viruses (AAVs), and Adenoviruses.

8. retroviruses
gag pol env
Simple RNA viruses that can integrate into the host genome.
They contain only three structural genes, which can be removed and replaced with the gene to be transferred
They engineered to render them incapable of replication and nontoxic to the cell,
the integrated DNA is stable and can accommodate up to 8 kb of added DNA, commodious enough for many genes that might be transferred.
A major limitation of many retroviral vectors, is that the target cell must undergo division for integration of the virus into the host DNA, limiting the use of such vectors in nondividing cells such as neurons.
In contrast, lentiviruses, the class of retroviruses that
includes HIV, are capable of DNA integration in nondividing cells, including neurons.
Lentiviruses have the additional advantage of not showing preferential integration into
any specific gene locus, thus reducing the chances of activating an oncogene in a large
gag – structural proteins
• pol – reverse transcriptase
• env – envelope proteinsnumber of cells.

9. adeno-associated viruses (AAVs), and adenoviruses.
AAVs do not elicit strong immunological responses, a great advantage that enhances the longevity of their expression
disadvantage is that the current AAV vectors can accommodate inserts of up to only 5 kb, which is smaller than many genes in their natural context.
adenovirus-derived vectors, can be obtained at high titer, will infect a wide variety of dividing or nondividing cell types, and can accommodate inserts of 30 to 35 kb.
However, in addition to other limitations, they have been associated with at least one death in a gene therapy trial through the elicitation of a strong immune response.

10. Targeting
To target viral vectors to specific cells or organ systems, it is usually necessary not only to add a targeting ligand to provide the new specificity, but also to disrupt pre-existing ligands (‘detargeting’) so that the new specificity replaces,
The new ligands should allow the virus to enter the cells by membrane fusion or active transport, through the same intracellular pathway as the native vector.
Type 5 adenoviruses, from which most human adeno vectors have been derived, bind to at least two molecules on their target cells – the Coxsackie adenovirus receptor (CAR) and cell- surface integrins (usually αvβ3 or αvβ5).
Domains on the adenoviral knob protein mediate binding.
Since the sequence and crysta structure of this protein is known, one can identify new ligand
sequences and incorporate them into positions that disrupt preexisting
patterns of binding and establish new ones.

12. Part II: Gene therapy of haemopoietic disorders
There are many potential applications for stem cells—from using stem cells to grow healthy tissues to studying stem cells to understand and treat birth defects and genetic diseases, to genetically manipulating stem cells for delivering genes in gene therapy approaches, to creating whole tissues in the laboratory using tissue engineering.
Centers for Disease Control’s National Center for Human Statistics indicates that approximately 3000 Americans die every day from diseases that may one day potentially be treated by stem cell technologies.
In this example, biodegradable
scaffolding materials were used to form the shape of the
bladder, and stem cells were seeded onto the scaffolding and
then differentiated to form bladder tissues.

13. HSC gene therapy timeline

14. There has been a rapid surge in clinical trials involving stem cell therapies over the last two to three years and those trials are establishing the clinical pathways for an emergent new medicine.
These early trials are showing roles for stem cells both in replacing damaged tissue as well as in providing extracellular factors that can promote endogenous cellular salvage and replenishment.

15. Gene therapy is effective in a number of monogenic diseases
1. Immunodeficiencies
-X-SCID immunodeficiency
- ADA- immunodeficiency
- chronic granulomatous diseases
2. Congential blindness:
Leber’s congenital amaurosis
3. Metabolic diseases - lipoprotein lipase deficiency
Some beneficial effects have been observed in treatment of:
Adrenoleukodystrophy
β-thalassemia

16. Severe combined immunodeficiency diseases
X-SCID
X-linked severe combined immunodeficiency (X-SCID) reflects the lack of the common γ-chain that is part of several interleukin (IL) receptors, which is required for T- cell development.
Affected patients have deficient T cells and natural killer cells and poorly functional B cells.
As recently reviewed,between 1990 and 2006, 20 individuals with X-SCID were treated in 2 gene therapy trials, 1 in Paris and 1 in London. All subjects lacked an HLA-identical donor.
X-Linked SCID which constitutes 40-50% of cases with the majority showing T– B+ lymphopenia.
– Mutations are found in the gene located on the X chromosome that codes for the -chain that is common to the receptors for IL-2, IL-4, IL-7, IL-9 and IL-15. thus the mutation impairs responses to a multitude of cytokines "Fig 17.2A”

17. Gene Therapy
Stem cells without correct γc gene
Retroviral vector with a correct γc gene

18. Seventeen of the 20 treated participants are alive and display nearly full correction of the T-cell deficiency by genetically modified T cells when evaluated between 5 and 12 years after the gene transfer procedure.
However, half of the trial participants remain on immunoglobulin replacement.
The natural killer cell deficiency also persisted. Older participants with hypomorphic mutations responded less well to the gene therapy procedure, possibly because of loss of thymic function with advancing age.
Unfortunately, 5 of the participants developed T-cell leukemia within 3 to 6 years after the gene transfer procedure.

19. Four were successfully treated with standard antileukemic therapy, and 1 died of refractory leukemia.
Vector integration analysis identified insertions near the LM02 protooncogene in 4 participants.
The malignancy was due to insertional mutagenesis: the retroviral vector inserted into the LMO2 locus, causing aberrant expression of the LMO2 mRNA, which encodes a component of a transcription factor complex that mediates hematopoietic development.

20. The initial clinical trials closed after the development of leukemia in the 5 participants.
Current-generation vectors are designed to avoid this mutagenic effect by using strategies such as including a self- inactivating or “suicide” gene cassette in the vector to eliminate clones of malignant cells

21. Severe combined immunodeficiency diseases
ADA-SCID
since 2000, 40 patients have been treated in Italy, the United Kingdom, and the United States.
CD34+ cells were transduced with a g-retroviral vector encoding the ADA gene.
Integration site analysis demonstrated vector insertions near protooncogenes, including LM02, but none of these patients developed leukemia.
Outcome unpredicted

22. Gene therapy of ADA deficiency
Retroviral vector containing correct ADA gene (cDNA) has been transduced into blood lymphocytes

24. Chronic granulomatous disease
Chronic granulomatous disease (CGD) is a rare inherited immunodeficiency characterized by recurrent bacterial and fungal infections due to a functional defect in the microbial-killing activity of phagocytic neutrophils.
It occurs as a result of mutations in genes encoding a multicomponent enzyme complex, the NADPH oxidase, that catalyses the respiratory burst.
The majority of patients have an X-linked form of the disease which is associated with mutations in a membrane-bound
component gp91phox.

26. all genes encoding for the subunits of the NADPH oxidase
have been cloned with CYBB being the first gene ever isolated
by positional cloning.2

27. Correction of neutrophil bactericidal function by overexpression of gp91phox subunit of NADPH oxidase

28. Induced Pluripotent Stem Cells
In 2006, Shinya Yamanaka of Kyoto University in Japan created the first iPSCs.
Yamanaka used retroviruses to deliver four transgenes Oct4, Sox2, c-myc,and Klf4 into mouse fibroblasts .
Expression of these four genes, which encode transcription factors involved in cell development, “reprogrammed” the fibroblasts back to an earlier stage of differentiation producing iPSCs.
In this approach somatic cells, such as skin
fibroblasts, would be taken from a patient in need of a transplant, and induced to form
differentiated cells of the organ of interest

29. IPSCs for Treating Sickle-Cell Disease
Rudolph Jaenisch and colleagues at Massachusetts Institute for Technology generated a great deal of excitement in 2007 with reports of the first therapeutic application of iPSCs.
Jaenisch and colleagues used iPSCs to correct sickle- cell anemia in a mouse homozygous for the human S- globin (sickle) allele
In this work, researchers used a humanized mouse model of sickle-cell anemia, so named because this is a mouse strain in which mouse globin genes were replaced by human genes (using a “knockin” approach), including the human S-globin allele
In 2006, Shinya Yamanaka of Kyoto
University in Japan created the first iPSCs.
retroviruses to deliver four transgenes Oct4, Sox2, c-myc,
and Klf4 into mouse fibroblasts (ST Figure 4–6(b)). Expression
of these four genes, which encode transcription
factors involved in cell development, “reprogrammed”
the fibroblasts back to an earlier stage of differentiation
producing iPSCs.

30. These humanized mice display many of the key symptoms, such as anemia, that occur in humans with sickle-cell disease.
Skin cells were isolated from the globin humanized adult mice, and the cells were reprogrammed using retroviruses encoding the four genes we described earlier when describing iPSCs: (Oct4, Sox2, c-myc, and Klf4).
Scientists then used homologous recombination to correct the iPSCs by replacing the S allele of the –globin gene with the A allele (the allele that does not cause disease).

31. The corrected iPSCs were differentiated in vitro to produce hematopoietic progenitor cells that were subsequently transplanted into anemic mice with the S alleles, which had been irradiated to destroy blood stem cells.
Transplanted iPSCs restored stem cells in the diseased mice and dramatically reduced symptoms of sickle-cell disease.
Although many barriers need to be addressed before such approaches are viable options for treating human diseases, this early-stage work has created great optimism about the potential of treating human diseases with iPSCs.hematopoietic

32. iPSCs for Treating Sickle-Cell Disease
In 2007, two different research groups demonstrated
that they had successfully derived patient-specific iPSCs by
reprogramming human skin cells taken directly from a volunteer
(ST Figure 4–6(c). One group reprogrammed skin
cells taken from the face of a 36-year-old woman and connective
tissue cells from the joints of a 69-year-old man, and
another group used skin cells from the foreskin of a newborn
boy. iPSCs were also recently produced without using
the c-MYC gene. This is a potentially important advance
because c-MYC is a known oncogene (cancer-causing gene).

33. Treatment of beta-thalassemia

34. Tumors correction
Gene therapy could be used to replace an inactive gene with an active one, or to neutralize an abnormal function gained by a mutated gene.
While the introduction of genetic material into haemopoietic cells, either to correct or to block a defect involved in malignant transformation, is appealing, it is also technically extremely challenging.
Not only is highly efficient transduction of target cells required, but also many of the mutations leading to cancer are effectively dominant, and simple introduction of a wild-type gene would not be of benefit.
Under these circumstances, the abnormal gene product must be silenced.

35. Several strategies for silencing are being evaluated, including antisense molecules that are oligonucleotides specifically designed to interfere with DNA or mRNA and prevent transcription or translation.
These challenges notwithstanding, several clinical and many preclinical studies have attempted to correct the function of haematological malignancies.
Synthetic oligonucleotides complementary to the junction transcripts of BCL–ABL fusion gene have been shown to block in vitro proliferation of Philadelphia positive leukaemia cells without impairing the growth capabilities of normal bone marrow progenitors.

36. Antisense-treated mice with severe combined immunodeficiency (SCID) injected with Philadelphia- positive leukaemia cells confirmed the capability of BCR-ABL antisense oligonucleotides to temporarily suppress the progression of the disease and to significantly enhance survival of the treated animals.

37. Leber’s congenital amaurosis – gene therapy
Most common cause of congenital blindness in children.
LCA2 – one of the forms – caused by mutation in the retinal pigment epithelium-specific 65-kD protein gene (RPE65).
RPE65 is required to keep light-sensing photoreceptor cells – the rodes and cones of the retina – in operating order.
The RPE65 gene encodes for the isomerohydrolase that isomerizes bleached all-trans-retinal into photosensitive 11-cis- retinal).
If no 11-cis-retinal is produced due to loss of or impaired RPE65 function, the chromophore rhodopsin cannot be assembled, and the photoreceptors remain insensitive to light stimuli.
LCA2 is a rare diseases – in USA only 2000 people but is untreatable and causes blindness early in life

39. Gene therapy of Wiskott-Aldrich syndrome
an X-linked, complex primary immunodeficiency disorder caused by mutations in the WAS gene; • characterized by recurrent infections, thrombocytopenia, eczema, autoimmunity, and an increased risk of lymphoma ; • The complex biology of this disease results from dysfunction in different leukocyte subsets, including defective T and B cell function, disturbed formation of the natural killer (NK) cell immunological synapse, and impaired migratory responses of all leukocyte subsets; • Severe WAS leads to early death because of infections, hemorrhage, or malignancy; • For these patients, the standard curative therapy consists of allogeneic hematopoietic stem cell transplantation (HSCT).
Wiskott-Aldrich Syndrome
– X-Linked disease showing a classic triad of symptoms:
bleeding diathesis "tendency to bleed" due to thrombocytopenia.
recurrent bacterial infections.
allergic reactions.
Longer term, patients have increased risk of developing malignancies, particularly of the lymphoid system.
The genetic basis of the disease is a mutation in the X-Linked gene coding for the Wiskott-Aldrich Syndrome protein "WASP", which is expressed in all hematopoietic stem cells.
Both T and B cells are functionally abnormal, patients are unable to respond to polysaccharide antigens.

40. Gene therapy of Wiskott-Aldrich syndrome

41. Gene therapy of Wiskott-Aldrich syndrome

43. Gene therapy of adrenoleukodystrophy
The efficacy of the HSC transduction was determined by assessing the number of hematopoietic cells expressing ALD protein in
bone marrow and peripheral blood cells by immunohistochemistry and the identification of identical insertion sites (IS) of lentiviral vector in myeloid and lymphoid cells from the same patient.

44. Gene therapy of adrenoleukodystrophy

45. Brain MRI Brain MRI analysis showed that progression of
demyelination was arrested 14 months after gene therapy in the first
patient and 20 months after gene therapy in the second patient.

46. Suicide gene therapy for graft-versus-host disease
Another successful application of gene therapy has been the transfer of a suicide gene into donor lymphocytes for the purposes of controlling allo-reactivity in the context of allogeneic hematopoietic stem cell transplantation.
The basic concept is to insert a gene that renders the target susceptible to drug-induced cell death.
More than 100 patients have been treated worldwide to date in several phase 1 and 2 studies. Plans are underway for a phase 3 study with industry support.

47. A number of genetic systems have been used or explored experimentally.
Two have been tested in clinical trials:
With a vector having the thymidine kinase gene, and post treatment is with ganciclovir.
The thymidine kinase gene from Herpes simplex virus
The other system involves caspase-9.69 T cells containing this gene can be abrogated using a dimerizing drug.

48. GVHD) by suicide gene therapy
GVHD) by suicide gene therapy. The allogeneic donor serves as a source of lymphocytes that are genetically modified by introducing the suicide gene, which has the potential to convert a prodrug into an active drug.
As shown on the right, this provides an opportunity for beneficial graft-versusinfection and graft-versus-leukemia affects. If GVHD occurs, administration of the prodrug results in ablation of allo-reactive lymphocytes and control of the disease.
By the time the T cells are ablated, engraftment has been established and immunity is generally sufficient to protect from infections.

49. The FDA approved the California-based biotechnology company Geron Corporation for the first U.S. clinical trial to use HESCs to treat individuals with spinal cord injuries.
Although much work remains to be done in the spinal cord repair and regeneration field, researchers are optimistic that neural stem cell transplants may be ready for human clinical trials in the next three to five years, offering hope to the many individuals who are affected by spinal cord injuries.
FDA hasn’t approved any human gene therapy product for sale.

50. Spinal Cord Injury http://www.geron.com/ GRNOPC1 Trial/

51. GRNOPC1 Induces Remyelination after Spinal Cord Lesions in Rodents
Geron Oligodendrocyte Progenitor Cells GRNOPC1

52. GRNOPC1 Improves Locomotors Behavior after Spinal Cord Injury

53. GRNOPC1 Promotes Neural Outgrowth

54. Ethical Issues Involving Stem Cells
Clergy, politicians, researchers, and the general public continue to passionately debate the merits of stem cells.
One issue at the root of these debates is the source of human stem cells, in particular hESCs, and their potential uses and abuses.
Although international guidelines on ethical use of stem cells have been established, there is currently no international policy governing stem cell use.
In the United States, the FDA has rules regarding the purity of stem cells and their applications in clinical trials and medical products.

55. On August 9, 2001, President George W
On August 9, 2001, President George W. Bush announced a ban on using federal funds to create an embryo for the purpose of isolating embryonic stem cells.
Many private foundations are providing hundreds of millions of dollars for stem cell researchers, and several states have enacted legislation to create stem cell research institutes.

56. References
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Cartier, N., & Aubourg, P. (2010). Hematopoietic Stem Cell Transplantation and Hematopoietic Stem Cell Gene Therapy in X‐Linked Adrenoleukodystrophy. Brain pathology, 20(4), ‏

57. Nienhuis, A. W. (2008). Development of gene therapy for blood disorders. Blood, 111(9), 4431-4444.‏
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