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CHAPTER 29 gene therapy describes the therapeutic gene-containing vectors being
directly injected into the patient (Fig. 29–1B). In the in vivo case, the
GENE THERAPY FOR gene is expressed, producing a therapeutic protein for treatment. The-
oretically, if the gene-modified cells are long-lived and able to expand
HEMATOLOGIC DISEASES inside the body, a single gene therapy can be sufficient to provide a life-
long therapeutic effect. Current gene therapy technologies have reached
the point that many types of single-gene hematologic deficiency diseases
can be permanently corrected, for example, X-linked severe combined
Hua Fung and Stanton Gerson immunodeficiency (X-SCID) and adenosine deaminase deficiency
severe combined immunodeficiency (ADA-SCID).
Gene therapy has improved significantly in effectiveness since
the mid-1980s when the first experiment using stem cell gene transfer
SUMMARY was successful. Scientists initially encountered several serious obsta-
cles. First, there was difficulty in delivering a modified gene into HSCs
The term gene therapy describes treatment resulting from expression of a because of their lack of cell-surface receptors and their quiescent state.
1
transferred gene (or transgene) in diseased or other cells by engineered Second, early X-SCID gene therapy was interrupted because 20 percent
vectors. Once within the cell, the transgene can direct synthesis of a therapeu- of patients developed a T-cell type of leukemia within 3 to 6 years after
tic protein that can complement a genetic deficiency or confer upon the cell a therapy. This event was caused by gene therapy-related viral vector inser-
desired phenotype or function. Many clinical trials have involved gene therapy tional mutagenesis; the vector contained powerful enhancer elements
for patients with various gene-deficient hematologic diseases, such as severe within its long terminal repeats (LTRs) that inserted close to, and acti-
2
combined immunodeficiency, hemophilia, Wiskott-Aldrich syndrome, chronic vated the LMO2 protooncogene. Over 3 decades, these problems have
granulomatous disease, aplastic anemia, hemoglobinopathies, HIV infection, been (for the most part) resolved. With an array of cytokine stimula-
tion cocktails to improve HSC receptivity to engineering, and improved
and leukemia. Results from some clinical trials indicate that gene therapy lentiviral vectors, the HSC transduction rate in humans can reach
can cure or improve many inherited or acquired hematologic disorders. This 80 to 100 percent. In addition, myeloablative conditioning regimens
3,4
chapter reviews the basic principles of gene transfer and the results of selected (e.g., busulfan, melphalan and 1,3-bis-[2-chloroethyl]-1-nitrosourea
preclinical and clinical studies. [BCNU; aka carmustine]) that decrease the number of endogenous
stem cells prior to infusion of the engineered HSCs has proven to be an
effective method to increase engraftment rates (average: 1 to 2 copies
of gene marking/cell). High-level stem cell engraftment is a critical
3,4
DEFINITION AND HISTORY factor for the gene therapy of chronic granulomatous disease, X-linked
adrenoleukodystrophy (X-ALD) and metachromatic leukodystrophy.
3–6
Gene therapy is a promising treatment for several inherited or acquired Moreover, newer, safer self-inactivating (SIN) viral vectors have been
hematologic disorders. Gene therapy involves the introduction of developed in which viral LTR enhancers are completely removed. Using
a functional gene to replace a mutated gene or a therapeutic gene to these newer vectors, no patient has developed therapy-related malig-
provide a missing or defective protein to the organism. In some cases, nancy in several clinical trials, some of which have followed patients
the patient’s blood cells are removed and special, targeted cells such for as long as 8 years. Gene therapy is no longer a hypothetical form
3,4
as hematopoietic stem cells (HSCs) are selected for engineering. The of therapy; some trials have achieved clinical correction for as long as
therapeutic genes are introduced into a vector and delivered into the 12 years. Gene therapy has, thus, undergone a renaissance. This chapter
targeted cells. These targeted, gene-modified cells are reinfused back discusses two critical technical factors in gene therapy: targeted cells
into the patient. Because this method modifies cells outside the patient’s and delivered vectors; briefly reviews the gene therapy of several com-
body, it is called ex vivo gene therapy (Fig. 29–1A). By contrast in vivo mon hematopoietic genetic deficient diseases; and describes new strate-
gies of in vivo selection and insertion site-targeted gene therapy.

Acronyms and Abbreviations: AAV, adeno-associated virus; ADA-SCID, adenosine GENE THERAPY TARGETED CELLS
deaminase deficiency severe combined immunodeficiency; ARSA, arylsulfatase A;
BCNU, 1,3-bis-(2-chloroethyl)-1-nitrosourea; CAR, chimeric antigen receptor; CCR5, HEMATOPOIETIC STEM CELLS
chemokine (C-C motif) receptor 5 gene; CGD, chronic granulomatous disease; CLL, HSCs are the cell of choice for many gene therapy applications for several
chronic lymphocytic leukemia; CRISPR, clustered, regularly interspaced, short palin- reasons. First, many blood cell disorders originate at the stem cell level
dromic repeats; DSB, double-stranded break; FA, Fanconi anemia; FVIII, factor VIII; and, therefore, gene-corrected HSCs are the best candidate for replace-
FIX, factor IX; GCV, ganciclovir; GVHD, graft-versus-host disease; Hgb, hemoglobin; ment. Second, HSCs are a long-lived and self-renewing population and
HR, homologous recombination; HSC, hematopoietic stem cell; HSV, herpes simplex may reduce or eliminate the need for repeated administration of gene
virus; HSV-TK, herpes simplex virus thymidine kinase; iCasp9, inducible caspase 9 therapy. HSCs are readily obtained in the blood, marrow, or umbilical
protein; IL2RG, interleukin-2 receptor gene; LTRs, the long terminal repeats; MGMT, cord blood. They are also easily selected and manipulated in the labora-
O -methylguanine-DNA methyltransferase; MLD, metachromatic leukodystrophy; tory and can be returned to patients relatively easily. Third, engineered
6
SIN, self-inactivating; siRNA, small interfering RNA; TALEN, transcription activator- HSCs are able to correct defects in all hematopoietic lineages. Fourth,
like effector nuclease; TMZ, temozolomide; WAS, Wiskott-Aldrich syndrome; X-ALD, HSCs migrate to several tissues in the body—primarily the marrow, but
X-linked adrenoleukodystrophy; X-SCID, X-linked severe combined immunodefi- also the liver, spleen, and lymph nodes. These may be strategic locations
ciency; ZFN, zinc-finger nuclease. for localized delivery of therapeutic agents for disorders unrelated to the
hematopoietic system, such as for patients with liver diseases.

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