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442 Part V: Therapeutic Principles Chapter 29: Gene Therapy for Hematologic Diseases 443

is sufficient to markedly reduce the bleeding risk. A new improved enhance conventional chemotherapy by protecting the marrow cells
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AAV vector (AAV8) was used. This vector has a self-complementary through gene transfer. Chemotherapy has a limited therapeutic win-
genome to improve transduction efficiency, and was designed to pro- dow because of its severe toxic effect on marrow cells, and its leuke-
duce fivefold higher levels of capsid protein to reduce a potential cyto- mogenic potential. Because the lethal effect of chemotherapy is mainly
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toxic T-cell response and increase liver tropism. The AAV vector is not DNA damage, especially methylating O -guanine, to overcome the side
genome-integrating and maintains itself as an intracellular episome. Its effects, a strong chemoresistant DNA repair gene, a mutant (P140K) of
gene expression in growing cells is transient because episomes may be MGMT was introduced into brain tumor patients’ autologous HSC by
lost with each cell division. But in quiescent tissues the AAV vector is a γ-retroviral vector, thus the patient’s transduced marrow progenitor
capable of mediating long-term gene expression as episomal chroma- cells could be protected by the modified MGMT, permitting them to
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tin. A single intravenous infusion of the vector was given to six adult tolerate more cycles of chemotherapy. A phase I clinical trial demon-
male hemophilia B patients who had been treated with recombinant FIX strated that intensification of chemotherapy was feasible, and there was
for many years. No notable acute or chronic toxicities were observed. an improvement in therapy outcome and patient survival in these small
All six patients displayed stable FIX expression at 2 to 11 percent of studies. 52,53 A similar result was also observed in a recent clinical trial
normal blood levels for 3 years. Four of these patients discontinued with a lentiviral vector (reported at the ASH meeting 2014).
recombinant FIX treatment and remained free of spontaneous hemor-
rhage. 15,43,45 The same research team is attempting a similar approach for
hemophilia A gene therapy; however, FVIII gene expression has been NEW TECHNOLOGIES USED IN
inefficient. One reason is gene size. The FVIII coding sequence is 7 kb, GENE THERAPY
which far exceeds the normal packaging capacity of AAV vectors. By
modifying the B domain, the FVIII size was reduced to a 5.2-kb AAV IN VIVO SELECTION AND
expression cassette, which is more efficiently packaged. Also, a hybrid
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liver-specific promoter was introduced into the vector. The resulting O -METHYLGUANINE-DNA
new AAV vector has shown high (15 percent of normal) FVIII expres- METHYLTRANSFERASE SELECTIVE METHOD
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sion for 20 to 45 weeks in four macaques. This AAV vector will be used Whether stem cell gene therapy can cure a disease is dependent on the
in a trial of hemophilia A gene therapy in the near future. functionality of the gene-corrected cell. In the early successful gene
therapy trials for X-SCID and ADA-SCID, a low (0.1 to 1.0 percent)
engraftment rate was sufficient for long-term gene correction, and the
FANCONI ANEMIA single most important factor was that the transgene conferred a selec-
Fanconi anemia (FA) is caused by mutations in Fanconi genes, which tive survival advantage on the transgene-bearing cell. In contrast, a
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encode the DNA repair proteins that form a function complex main reason for poor responses in other gene therapy trials has been
(Chap. 35). FA cells are hypersensitive to DNA crosslinking agents. that the gene-corrected cells have no, or only a weak selective advantage,
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Sixteen Fanconi genes have been described. A defect in one of them will such that the gene-transduced cell levels are insufficient to provide a
lead to FA. The disease is characterized by a high risk of developing mar- clinical meaningful benefit. Therefore, in vivo selection is a key element
row failure and later myelodysplasia, acute leukemia, or cancers of other in the success of clinical gene therapy.
tissues. More than half of patients with FA are the result of FANCA The gene-corrected cells in the majority of hematologic genetic dis-
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gene mutations; therefore current gene therapy has focused on FANCA eases have no in vivo selection advantage. To overcome this problem,
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insufficiency. Gene therapy for FA is particularly challenging because of a second, selectable gene can be used to be coexpressed with the gene
the low numbers of HSCs in the stage of marrow failure, and FA cells that corrected the defect. The second gene turns the cells into selectable
are extremely sensitive to DNA damage when exposed to myelosuppres- cells. The selectable gene can be cloned into a vector along with cor-
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sive drugs used to condition the patient for stem cell transplantation. recting gene driven by a single or separate promoter. Experiments have
In a rare case, two identical twins had inherited FANCA mutations but identified several selectable genes including multidrug resistance pro-
with normal DNA repair in their blood stem cells. Functional FANCA tein, dihydrofolate reductase, and MGMT. MGMT shows the most
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in blood cells was found to be restored by a spontaneous intrauter- promising results in large animal and humans trials. MGMT encodes
ine self-correcting somatic mutation in a single HSC. The fact that a a DNA repair enzyme O -alkyguanine-DNA-alky-transferase, which
6
single HSC was sufficient to restore a fully normal blood system indi- confers resistance to the cytotoxicity of chemotherapy, such as BCNU
cates that FANCA gene therapy may require transduction of only a few and temozolomide (TMZ). A MGMT mutant, P140K-MGMT, has at
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HSCs. 48 least a 50-fold stronger effect on drug resistance resistance. P140K-
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In 2011, an international working group was established to facil- MGMT–expressed cells can be exposed to BCNU and TMZ selection
itate the development of gene therapy for FA. The initial protocol pressure (Fig. 29-4). Clinical trials have demonstrated that P140K-
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included delivery of a normal FANCA gene by a third-generation len- MGMT protects the gene-modified cells from TMZ-induced toxicity.
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tiviral vector into HSC and increasing HSC number by in vitro HSC This strategy has also been used in a mouse model of HIV gene therapy. 36
expansion using a combination of HOXB4 and DELTA-1 proteins. 50 Successful gene therapy with satisfactory gene marking levels
(approximately 30 percent) are dependent on three factors: a high
HSC transduction rate, a high engraftment rate, and in vivo selection.
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GENE THERAPY FOR CANCER Other trials indicate that if there are excellent transduction rates (80 to
Gene therapy for cancer has been widely exploited. A review was pub- 90 percent) and excellent engraftment rates (gene copy marking/genome
lished detailing the new developments in this field. This chapter has >0.5), these two factors can be sufficient to achieve a sustained gene
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described a few significant new approaches. correction, even without an in vivo selection mechanism. Nevertheless,
4
One of the most creative new approaches to cancer-targeted gene if there is a low transduction rate and low engraftment rate, MGMT-
therapy is the use of CAR for CLL, in which patient’s T cells were mod- mediated in vivo selection will be a very valuable tool to improve the
7,8
ified to target their own cancers (Chap. 92). Another strategy is to likelihood of successful gene therapy.

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