Williams Hematology



Gene Transfer: A Multistep Pathway

Inherited Genetic Disorders

Hematopoietic Disorders

Inherited Coagulation Deficiencies
Acquired Disorders

Acquired Immunodeficiency Syndrome

Neoplastic Disorders

Fetal and Neonatal Gene Therapy
Chapter References

Understanding vector cell–host interactions will guide the field of gene transfer. These complex interactions require the understanding of virology, cell biology, host response and immunology, and the disease processes. Initial enthusiasm for genetic correction of several inherited hematopoietic disorders has now been tempered by the lack of efficient gene transfer in several clinical studies. The development and application of vectors designed to infect hematopoietic cells has led to pseudotyping, chimeric vectors, and nonviral methods for gene repair.
The development of recombinant DNA technology during the past 20 years has created the potential that genetically defined diseases, whether hereditary or acquired, can be treated by genetic therapy. Human gene therapy consists of the insertion of a functioning gene into appropriate target cells of an affected individual to correct for a defective gene or to add a new function to these cells. This chapter reviews the basic principles of gene transfer and the results of selected clinical gene transfer studies.

Acronyms and abbreviations that appear in this chapter include: AAV, adeno-associated virus; ADA, adenosine deaminase; ENV, envelope protein; HbF, fetal hemoglobin; HIV, human immunodeficiency virus; HLA, human leukocyte antigen; IL, interleukin; LCR, locus control region; LTR, long terminal repeats; rAAV, recombinant adeno-associated virus; TIL, tumor infiltrating lymphocytes.

Effective genetic therapy requires a safe and efficient means of gene delivery. Viral vectors attempt to capitalize on the inherent efficiency of viruses to transfer and express their genetic information in eukaryotic cells. For gene therapy applications, vectors are designed that allow entry of the recombinant virus with subsequent integration into the cellular genome or maintenance as an episome without alteration of cell metabolism or production of new virus. Retroviruses, adenoviruses, and AAV are currently being developed for use in humans. Physical methods of DNA transfer widely used in the research laboratory are also being considered for therapeutic applications. (For the definition of vector and other terms used in this chapter, see Table 19-1.)


Typically, viruses bind and enter host cells via receptor-mediated endocytosis (Fig. 19-1). During productive infection, the viral genome is transported to the nucleus, where it usurps host cellular machinery to direct the synthesis of viral-specific proteins and assembly of new viral particles. This complex process is shown in Fig. 19-1. Virus entry can be accomplished by binding to cell surface receptors or through nonspecific attachment. A list of cell receptors for specific viral vectors is shown in Table 19-2.

FIGURE 19-1 Vector–target cell interaction. Schematic representation of recombinant virus binding and internalizing through the cell membrane with release of the vector genome from the endosome. Following vector genome trafficking to the nucleus, episomal or integrated vector species allow for transcription of the vector transgene and translation into a functional protein.


Upon binding and entry into the target cell, vector nucleic acid must be modified and transported to the nucleus. Retroviral vectors carry reverse transcriptase and convert their single-stranded RNA genomes to double-stranded DNA. The preintegration nuclear protein complex of oncoretroviruses, such as murine leukemia virus, is relatively unstable and cannot traverse the nuclear membrane. Therefore, such vectors are able to integrate their genome in dividing cells. In contrast, the preintegration complex of lentivirus, such as those based on HIV, is relatively stable and transported through the intact nuclear membrane.1 Lentiviral vectors have a greater capacity to integrate their genome into quiescent cells. Both oncoretroviral and lentiviral vectors integrate into the host cell genome randomly. Random insertion may allow for inhibition of transgene expression due to transcriptional silencing.2
DNA vectors such as AAV or adenovirus behave differently. The rAAV genome is composed of single-stranded DNA molecules flanked by palindromic inverted terminal repeats. Upon entry into the nucleus, the single-stranded genome must be converted into a double-stranded form before the encoded gene can be transcribed. The double-stranded genome may remain episomal, it may form large tandem concatemers, or the genome may integrate into either single- or multicopy configuration. Persistent gene expression can be achieved in both episomal and integrated states. The double-stranded DNA genome of adenoviral vectors remains episomal, and therefore these vectors are useful for achieving transient expression in a proliferating cell population.
Host immunologic responses to viral capsid or coat proteins limit infection or readministration of virus. For example, the cell-mediated immune response directed against adenoviral coat proteins produces a significant inflammatory response.3 Host response to the transgene protein product is dependent on the antigenicity of the expressed product. The expression of a new transgene protein may trigger cell- and humoral-mediated immune responses.
Retroviral Vectors
Retroviral vectors for gene transfer studies are derived from the murine leukemia virus family. Figure 19-2 outlines the integrated proviral form of the Moloney murine leukemia virus. The genome is flanked by LTRs that contain the transcriptional control elements, polyadenylation signals, and sequences required for replication and integration.4,5 The viral genome contains coding sequences for structural proteins (GAG), reverse transcriptase (POL) and other viral enzymes, and ENV. A packaging signal, located at the 5′ end of the coding sequence y, facilitates incorporation of RNA into the capsid. Retroviruses capable of infecting murine cells and a few rat cell lines are described as having ecotropic specificity, which is based on interaction of the viral ENV (gp70) with a defined transmembrane protein that acts as a receptor.6,7 Retroviruses that have a broader host range that includes human cells are termed amphotropic; the cellular receptor for amphotropic viruses has been identified as an anion transport protein. Retroviral amphotropic receptor is expressed at very low levels on murine and human stem cells.8 Limiting amounts of virus receptor and the requirement for cell cycling explain the low transduction of human stem/progenitor cells. To enhance virus binding, amphotropic or ecotropic retroviral ENV can be substituted (pseudotyped) with ENV from other viruses, such as gibbon ape leukemia virus9 or the rhabdovirus vesicular stomatitis virus.10 Virus pseudotyping confers the receptor specificity of the ENV and improves the susceptibility of target cells to the new hybrid virus.11

FIGURE 19-2 Schematic representation of the Moloney murine leukemia proviral form. GAG, virion structural protein; POL, reverse transcriptase/ integrase; ENV, envelope glycoprotein; LTR, viral RNA synthesis, integration; y, encapsidation.

Packaging cell lines have been engineered to produce retroviral vectors carrying genes of interest without the production of replication-competent virus (see Fig. 19-2). Such cell lines express a “helper” genome or genomes encoding the viral structural proteins, while a separate proviral genome contains the gene to be transferred and retains the cis-active elements required for genome encapsidation, replication, and integration.12,13 Typically, 106 to 107 infectious vector particles per milliliter of culture are necessary to achieve gene transfer into target hematopoietic cells. Pseudotyped retrovirus can withstand ultracentrifugation, and concentrated virus (>109 particles per milliliter) can be prepared. Retroviral gene transduction requires that target cells be actively replicating; quiescent (G0) cells are refractory to proviral integration.14,15 Pseudotyped lentiviral vectors offer a potential solution to the two problems of low viral receptor expression and quiescent human stem cells.
Replication-competent (wild-type or helper) virus contamination of retroviral vector stocks must also be eliminated. Studies showed that lethally irradiated rhesus monkeys developed thymic lymphomas following autologous transplantation of marrow cells that had been transduced with a vector preparation contaminated with replication-competent virus.16 Multiple copies of a proviral genome or genomes were detected in the tumor cell DNA, implicating insertional mutagenesis as a mechanism of carcinogenesis. To prevent the production of replication-competent virus by recombination between vector and helper genomes, areas of homology between the two have been eliminated. Mutations have been introduced into residual viral coding sequences in the vector genome, and/or the structural genes required for helper function have been separated into two transcriptional units. Newer packaging lines have virtually eliminated detectable production of replication-competent virus. Similar packaging strategies employed for HIV-based vectors are paramount to avoid any possibility of wild-type HIV production.
Adeno-associated viruses are single-stranded DNA parvoviruses that require helper virus proteins (adenovirus or herpesvirus proteins) for replication and subsequent viral production in permissive cells. In the absence of helper virus, AAVs can persist in host cell genomic DNA as a multi-molecular concatenate and as an episome.17 Some or all of these molecular species can be detected, depending on the cell or tissue type transduced. Unlike other potential viral vectors, wild-type AAV is unique with respect to its site-specific integration into a particular region on chromosome 19.18 Viral integration has no apparent effect on cell growth or morphology. Adeno-associated virus has a broad host range through its use of heparan sulfate as an attachment molecule; virtually every mammalian cell line can be productively or latently infected. Despite its wide host range, no disease has been associated with AAVs in human or animal populations.
Recombinant AAV production relies on the cotransfection of helper and vector plasmids into a permissive cell line. Concomitant infection with adenovirus for AAV replication provides the necessary complementing functions (Fig. 19-3). Contaminating adenovirus can be removed by gradient centrifugation or inactivated by heating; adenovirus is heat sensitive, but AAV is heat resistant. Advances in rAAV preparation have eliminated the production of adenovirus, and rapid purification can be performed using heparan-affinity chromatography. Recombinant AAV purification now yields titers of 1012 to 1014 particles. The ability to produce high-titer virus, along with its tropism for muscle, liver, and central nervous system, has broadened the appeal of this vector.19 Recombinant AAV vectors used in a phase I clinical trial for cystic fibrosis patients indicate that they are safe20 (Fig. 19-4).

FIGURE 19-3 Generation of recombinant RNA retroviral vectors. The diagram depicts the strategy to generate amphotropic recombinant virus containing a gene of interest (hatched area). Plasmid DNA containing the recombinant proviral genome of interest and the retroviral y (packaging) region is introduced into a packaging cell containing an integrated wild-type helper genome lacking the y region. Integration of the vector provirus allows for the stable production of retroviral particles. Clones identified as producing the highest concentration of vector particles are subsequently used to generate virus for gene transfer studies.

FIGURE 19-4 Recombinant DNA AAV production. A permissive cell line is cotransfected with helper and recombinant plasmids and infected with adenovirus. Both adenovirus and the helper plasmid (which produces the REP and CAP proteins) are necessary for rAAV replication and packaging. Due to the lack of homology between the two plasmids, no wild-type AAV is generated. Recombinant virion formation occurs in the cell nucleus, and both rAAV and adenovirus are released following cell lysis. Adenovirus is either inactivated by heating or removed by density centrifugation.

Several nonviral methods have been developed to permit target cells to take up DNA. Originally, these methods allowed for nonspecific binding and entry of DNA, either as calcium-phosphate complexes or liposomes or by direct microinjection of DNA into single cells.21 These methods appear to be less efficient in gene transduction than are viral vectors, and gene expression is transient. Transduction of long-term repopulating stem cells using these methods has not yet been achieved.
The use of RNA or DNA oligonucleotides to repair mutations or alter RNA splicing mutations has gained interest. Hybrid DNA-RNA oligonucleotides designed to repair the single base change responsible for sickle cell disease have achieved correction of the mutation in cell lines.22 Oligonucleotide targeting of mutant stem cells could generate permanently corrected progeny without the need for permanent transgene insertion. Oligo-based correction of aberrant RNA splicing in thalassemic cells has been described.23
Mammalian artificial chromosomes, suitable for large cDNAs or large genomic sequence capacity, are currently being tested. Mammalian artificial chromosomes include genetic elements that allow for their own replication. These self-replicating artificial chromosomes exist as episomes. Strategies to overcome limited mammalian artificial chromosome introduction into cells are under investigation.24
Correction or treatment of genetic disorders affecting marrow is likely to be based on gene transfer into hematopoietic stem cells. If successfully targeted for gene transfer, stem cells would ensure the continuous production of genetically modified hematopoietic cells over the lifetime of the patient. In general, the more mature hematopoietic cells are inappropriate targets for gene transfer due to the lack of self-renewal and long-term survival. An important exception is lymphocytes, which have a long life span.
Stem cells can be highly purified based on their immunophenotype, including an absence of lineage-specific markers and the class II histocompatibility antigen HLA-DR and the presence of certain markers, such as CD34 and Thy1 (see Chap. 141). Cells having the stem cell immunophenotype that contributes to marrow repopulation are found in the blood after cytotoxic drug and/or cytokine administration. CD34+ mobilized peripheral blood, marrow, and umbilical cord blood stem cells are appropriate for therapeutic gene transfer. More primitive CD34+/CD38– cells, which maintain a greater repopulating potential, are resistant to Moloney-based retroviral transduction. The identification of CD34–25 and fetal neuronal cells26 capable of multilineage bone marrow reconstitution are new targets for hematopoietic gene transfer.
Efforts have been focused on enhancing the efficiency of retroviral-mediated gene transfer into hematopoietic stem cells. Cytokine-mobilized peripheral blood stem cells, cytokine combinations (e.g., stem cell factor, IL-3, IL-6, Flt3-ligand, and thrombopoietin), recombinant fibronectin support, and pseudotyping or stromal cells have been used to sustain stem cells during vector exposure in vitro and to enhance the probability of cell division required to achieve integration of the proviral genome.27 Sixty to eighty percent of murine repopulating stem cells, and ten to twenty percent of the primate stem cells are susceptible to retroviral-mediated gene transfer under current conditions of in vitro culture during exposure to virus.28 Alternative target cells for therapeutic gene transfer are circulating T lymphocytes. Such cells can be induced to divide many times in vitro, with a resulting several-fold amplification of the initial cell population. Interleukin-2 and an anti–T-cell-receptor antibody synergistically act as mitogens during in vitro culture.
ADA deficiency (see Chap. 88) best exemplifies the current paradigm for gene replacement therapy. Even with less than 5 percent of normal ADA levels, patients usually retain normal immune function. Retroviral transduction of the normal ADA gene into blood T lymphocytes of affected patients corrected the deficiency phenotype and suggested the feasibility of a gene transfer approach.29 Results of transfer of the ADA gene into blood lymphocytes of several patients who did not have a suitable marrow donor and did not respond well to enzyme administration have been very encouraging.30
The introduction of the ADA gene into pluripotential hematopoietic stem cells is a strategy for achieving a permanent cure of ADA deficiency.31 This has the advantage of generating gene-corrected T cells with a full immune repertoire, whereas T-cell–targeted gene transfer is likely to correct cells of an already determined antigen specificity. Amplification of the gene-corrected T cell population is hypothesized to provide a selective growth advantage, despite the low efficiency of gene transfer into stem cells. This was tested using stem cells derived from umbilical cord blood harvested from ADA-affected newborns.32 Four years after three newborns were given infusions of transduced autologous umbilical cord blood CD34+ cells, the frequency of gene-marked peripheral T lymphocytes remained at a level of 1 to 10 percent. On cessation of ADA replacement enzyme, a decline in immune function occurred in one patient despite evidence of gene transfer.
High-level regulated globin gene expression is required for therapy of severely affected patients with sickle cell disease and b thalassemia.33 Introduction of a normal b-globin gene into hematopoietic cells should be useful for correcting the defect in homozygous b thalassemia. In sickle cell disease, both the increased expression of a normal globin gene and a decrease in the amount of the bS chain in mature red cells are required to be therapeutically effective. Increased fetal hemoglobin production ameliorates the severity of sickle cell disease and thalassemia. Production of g-globin chains leading to accumulation of HbF (a2g2) in erythroid cells may therefore be therapeutic. This result might be achieved by introduction of genetic information that alters the pattern of globin gene expression.
Retroviral vectors have been used to transfer the b-globin gene into murine hematopoietic stem cells. The human b-globin gene was expressed in the erythroid cells of most animals for long periods but only at a level of 1 to 2 percent of normal murine b-globin levels.34 Regulatory sequences upstream from the globin gene cluster, collectively termed the LCR, offer new hope for the design of therapeutic vectors.35 Globin genes linked to LCR elements exhibit high-level expression in transgenic mouse models and erythroleukemia cell lines.36 Producer clones for most vectors that have LCR elements have low titers, however, and these vectors are prone to rearrangement on insertion into target hematopoietic cells. Retroviral vectors utilizing truncated LCR elements linked to a human b-globin gene have produced levels of expression equivalent to that of endogenous genes in both erythroleukemia cell lines and murine hematopoietic cells.37 Retroviral vectors have been modified extensively to eliminate sequences prone to produce viral rearrangement.38 The use of a-globin LCR elements linked to b- or g-globin genes is an alternative approach.39
Transfer and regulated high-level expression of a human globin gene linked to required transcriptional control elements have been achieved in erythroleukemia cells with an rAAV vector.40 Primary hematopoietic progenitors may be transduced, but receptor levels may vary among individuals. Stable integration may not occur in all cells that initially take up and express the rAAV genome. Differences in AAV transduction of CD34+ cells are the result of variable heparan sulfate surface expression. Modification of rAAVs to facilitate binding to cells lacking receptor has been described.41
Gaucher disease is one of several lysosomal storage disorders (see Chap. 79) in which gene transfer to hematopoietic stem cells is potentially therapeutic. Phenotypic correction of patient fibroblasts and lymphocytes has been demonstrated following retroviral-mediated transfer of the glucocerebrosidase gene.42 Transduction of murine hematopoietic stem cells has been achieved, with long-term expression of the glucocerebrosidase gene in macrophages of transplanted mice.43 Expression in human hematopoietic cells has also been described after transduction of progenitors and cells capable of initiating long-term marrow cultures.44 Two clinical trials using Moloney-based retroviral vectors targeted to CD34+ cells yield similar results with low transduction of peripheral blood cells.45,46
Phenotypic correction of phox 91 and phox 47 knockout mice was produced following bone marrow retroviral transduction.47,48 Following treatment, knockout mice resisted bacterial and fungal infection due to a functional NADPH oxidase. These results led to clinical trials for phox-47-deficient CGD patients who received Moloney retroviral–based transduced mobilized peripheral blood.49 Superoxide generation in blood granulocytes of patients could be detected in the peripheral blood for several weeks, suggesting that long-term stem cells were not transduced. Since mothers heterozygous for the X-linked form of the disease who have only 10 percent of normal granulocytes are clinically normal, long-term correction of only a small percentage of cells should result in therapeutic benefit.
The Fanconi anemia complementing genes A and C (FANCA and FANCC) have been genetically engineered into retroviral and rAAV viral vectors.50,51 Phenotypic correction of Fanconi anemia lymphoblastoid cell lines was demonstrated by cell growth in the presence of clastogenic agents to which Fanconi anemia cells are typically sensitive and by reduced susceptibility to chromosomal breakage. Gene-corrected stem cells should have a selective growth advantage when transplanted into patients. These vectors were used to transduce purified hematopoietic progenitors from patients carrying defective FANCA and FANCC alleles. Retroviral-mediated gene transfer performed in four FANCC patients indicated gene marking of blood cells, improved clonogenic growth, and transient improvement in bone marrow cellularity and blood counts.
Sustained therapeutic levels of factors VIII and IX would significantly affect the clinical course for the hemophilias. Transfer of factors VIII and IX into hemophilic A and B animals has been established.52,53 Adenoviral vectors carrying the factor VIII or IX cDNAs produce high levels of circulating factor. However, due to the elimination of the virus by the host immune response, factor production lasts for only a period of weeks.
The use of rAAVs for hemophilia is promising. A single intramuscular or intravascular injection of vector produces sustained long-term factor IX expression (>1.5 years) in mice54 and hemophilia B canines.55,56 Targeting to either skeletal muscle or liver in hemophilic B canines produces 1 to 3 percent of normal factor IX levels. Unlike adenoviral vectors, toxicity is limited to adenoviral contamination and a modest humoral immune response to rAAV capsid.
Gene transfer approaches to treating HIV-1 infection have emerged. Because of HIV tropism for lymphocytes, gene targeting to hematopoietic stem cells has been proposed as a mechanism for achieving a desired therapeutic effect in differentiating T cells. Current gene transfer–based strategies for therapy of HIV are targeted at (1) elimination of infected cells, (2) interruption of viral replication, or (3) inhibition of cellular infection.57 Strategies designed to interfere with HIV receptor binding and cellular entry are currently being explored.
Graft-versus-host disease associated with the therapeutic infusion of donor lymphocytes after allogeneic bone marrow transplantation can be effectively controlled by the expression of herpes simplex virus thymidine kinase suicide gene into the allogeneic lymphocytes.58 This was achieved by the selective elimination of transduced lymphocytes by ganciclovir infusion. This clinical trial has demonstrated the best evidence to date for effective gene transfer.
Various strategies have been proposed to enhance the immune response to neoplastic cells for therapeutic benefit.59 TIL cells may serve as vehicles to carry tumor-inhibitory or immune-enhancing cytokines, such as tumor necrosis factor (TNF) or IL-2, into tumors after retroviral-mediated gene transfer in vitro. Another strategy attempts to augment cytotoxic T cell and/or natural killer cell response to tumor antigens by expression of cytokine or HLA genes in neoplastic cells. Explanted tumor cells are transduced in vitro and reimplanted subcutaneously into the patient from whom they were obtained. Substantial data from murine models support this approach.60 Initial phase I clinical trials provided evidence for some resolution of tumors. Current efforts using gene-transduced tumor vaccines is directed at optimizing dendritic cell activation by cytokine genes and overexpressing relevant tumor-associated peptides.61
Hematopoietic toxicity associated with intensive chemotherapy is frequently dose limiting. Gene transfer into stem cells of patients undergoing autologous transplantation might be used to create marrow resistant to subsequently administered chemotherapy. This concept has been tested in oncology patients using a retroviral vector that confers multidrug resistance through expression of P glycoprotein (gp140).62 Patients receiving chemotherapy for a variety of solid malignancies exhibited no significant hematopoietic protection.63,64 These results highlight the low transduction efficiency of stem/progenitor cells. Overexpression of mdr-1 produces a myelodysplastic marrow in mice, suggesting that alternative dominant selectable agents be employed.
Advances in prenatal diagnosis and fetoscopic technique now allow for the transfer of genetic material in utero. Introduction of adenoviral vectors into the fetal airway or umbilical vein of neonatal sheep expressed marker and therapeutic genes in many organs.65 Antiadenoviral immunologic reactions limited transgene expression in late-stage gestation. Neonatal gene transfer using rAAVs led to widespread correction of pathology in a murine model of lysosomal storage.66 Risks of in utero gene therapy apply to the mother, the fetus, and future generations, given the potential for vector insertion into the germ line. Currently there are insufficient safety data to support a phase I trial.67

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Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn
Williams Hematology


5 comments on “CHAPTER 19 GENE THERAPY

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  2. […] Stem Cell Blog – The role of hematopoietic stem cell transplantation in treatment of radiation exposureStem Cell ApheresisThe benefits of canine stem cell researchWhat is the immune response to stem cell transplantsStem cell research tried on different conditionsStem Cell BlogBone Marrow Transplant Compared to Mobilized Peripheral BloodGood News/Bad News on Compensating Bone Marrow DonorsMeeting CordlifeCHAPTER 19 GENE THERAPY […]

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