Stem Cell Gene Therapy

Chapter 77

Stem Cell Gene Therapy Brian R. Davis* and Nicole L. Prokopishyn** *

The University of Texas Health Science Center at Houston, Houston, TX, USA, **Gene Editing, Institute for Inherited Disease Research, Newtown, PA, USA

Chapter Outline Introduction Gene Addition Viral Vectors Nonviral Integration Strategies Synthetic Microchromosomes Genome Editing RNAeDNA Hybrids Single-Stranded DNA Oligonucleotides Small Fragment Homologous Replacement (SFHR) Triplex-Forming Oligonucleotides (TFOs) Adeno-associated Virus Vectors Requirements for Successful Stem Cell Gene Therapy Genetic Modification Directly in Stem Cells Gene Addition Genome Editing Genetically Corrected Stem Cells and Their Relevant Differentiated Progeny Consistently Present at Sufficient Frequency In Vivo Gene Addition Genome Editing Physiologically Appropriate Expression Levels of the Corrected Gene Product in the Relevant Cells

937 939 939 939 939 939 940 940 940 940 940 941 941 941 941

942 942 942

Gene Addition Genome Editing Absence of Interference from Endogenous Defective Gene Product on the Activity of the Corrected Gene Product Gene Addition Genome Editing Absence of Adverse Effects Gene Addition Genome Editing Genome Editing of Human Hematopoietic Stem/Progenitor Cells Microinjection-mediated Delivery of Macromolecules to Adult Stem Cells Genome Editing of the b-globin Gene in Hematopoietic Stem/Progenitor Cells RNAeDNA Hybrids Small Fragment Homologous Replacement Conclusion References

943 944

944 944 944 944 945 945 945 945 945 945 946 946 946

943

This volume surveys the biologic properties and potential clinical uses of various classes of adult stem cells. In this chapter, we focus specifically on ex vivo gene therapeutic modification of autologous stem cells from patients with genetic disease and discuss the elements crucial for successful clinical application of these therapies. In particular, we contrast gene addition and genome editing approaches for stem cell gene therapy, highlighting the particular challenges that each approach faces to achieve therapeutic benefit.

INTRODUCTION Primitive stem cells capable of self-renewing proliferation and single or multiple cell lineage progeny generation have been identified in several human and mouse tissues. For

example, various stem cells individually capable of producing hematopoietic, mesenchymal, endothelial, or liver cells have been identified in adult bone marrow. Although the biologic characterization of various nonhematopoietic stem cells is still in its early stages (e.g., extent of plasticity, ex vivo expansion, and cues in vitro and/or in vivo required to activate stem cells to produce progeny of a particular type), laboratory and therapeutic clinical experience with hematopoietic stem cells (HSCs) suggest that other stem cell types will likely have successful clinical application. The HSC has exhibited the ability to establish a normal, healthy blood system in patients following transplantation of normal blood stem cells from closely matched individuals e treating disorders of the blood system including immune deficiency, thalassemia, and

Handbook of Stem Cells, Two-Volume Set. DOI: http://dx.doi.org/10.1016/B978-0-12-385942-6.00077-9 Copyright Ó 2013 Elsevier Inc. All rights reserved.

937

938

VOLUME | 2

leukemia. HSC gene therapy offers significant promise for treatment of various hematopoietic diseases because genetic correction of autologous HSCs could result in long-term correction of blood system cells while avoiding the immune system complications resulting from nonidentical transplantation. For example, thalassemia and sickle cell anemia, the most common genetic diseases of blood, could potentially be treated either by delivery of the globin transgene to HSCs (provided that there is appropriate, erythroid-specific expression of the globin transgene in the progeny red blood cells) or by direct repair of a specific globin gene mutation in the HSCs (McCune et al., 1994; McInerney et al., 2000). Significant attention has been devoted to the isolation, culture, and genetic modification of human bone-marrowderived mesenchymal stem cells (MSCs), because they generate cells of cartilage, bone, adipose, marrow stroma, and possibly muscle (Bruder et al., 1997; Prockop, 1998; Pittenger et al., 1999; Gronthos et al., 2003). Provided that these genetically modified cells or their differentiated progeny can be efficiently delivered to the required tissues in vivo, genetic modification of these cells is a potential treatment for various genetic diseases affecting mesenchymal cells such as osteogenesis imperfecta, Marfan’s syndrome, and muscular dystrophy (Van Damme et al., 2002). Identification of somatic stem cells giving rise to liver (Peteren et al., 1999; Theise et al., 2000), pancreas (Ramiya et al., 2000), and brain (Johansson et al., 1999) raises the possibilities of future application of ex vivo stem cell gene therapy to treatment of diseases affecting these

Adult and fetal Stem Cells

organ systems. Furthermore, recent identification and isolation of multipotential adult progenitor cells (MAPCs) capable of significant ex vivo expansion and differentiation to multiple lineages including neurons, hepatocytes, and endothelial cells potentially makes these cells ideal targets for ex vivo stem cell gene therapy (Jiang et al., 2002; Reyes et al., 2002). Although one day it may be possible to specifically target various stem cells in vivo (e.g., based on a stem-cellspecific surface phenotype), this possibility is not available today. Instead, it is more likely that the relevant autologous stem cells will first be isolated from tissues of affected individuals and genetically modified ex vivo. The ability to isolate stem cells (hematopoietic and nonhematopoietic) from patients with genetic disease, genetically correct the stem cells, possibly expand them ex vivo, and transplant them back into patients with the goal of producing genetically corrected cells in vivo offers significant potential for the genetic treatment of human disease. Genetic modification in stem cells will be designed to either completely correct the genetic defect or at least compensate for the genetic defect. Gene defects occur in a variety of forms, ranging from a simple base pair mutation (e.g., resulting in an amino acid change, a frame shift, the introduction of a stop codon, or a splicing defect) to complete absence of a gene. Two general approaches can be used to correct defective genes within cells: gene addition and genome editing (outlined in Figure 77.1).

Defective Mutation Mutant protein produced

Endogenous gene Human chromosomal DNA (with mutation in gene)

Gene Addition

Introduced correct gene (with viral elements)

Endogenous gene Random insertion of correct gene (or gene segment) into chromosome Defective endogenous gene remains

Normal and mutant protien produced

Genome Editing Corrected sequence

Endogenous gene

Only normal protien produced

Endogenous gene corrected in the chromosome

FIGURE 77.1 Schematic comparison of gene addition and genome editing approaches to stem cell gene therapy.

Chapter | 77

Stem Cell Gene Therapy

GENE ADDITION Gene addition involves the delivery of corrective DNA (usually composed of the entire coding region of a gene and appropriate regulatory sequences) that compensates for or overrides the defective gene (Figure 77.1). The defective gene, unless it is completely absent, remains in the affected cells. This approach is particularly applicable when the endogenous gene product is not expressed (e.g., because of extensive deletion of the gene, a silencing mutation in the transcriptional regulatory sequences, or introduction of a stop codon near the start of the coding sequence). Successful application of stem cell gene therapy requires that therapeutic genes delivered to stem cells persist in the self-renewing stem cells and in their mature and differentiated progeny. This requirement for transgene persistence is critical because (1) transgenes present only transiently in stem cells undergoing self-renewing proliferation will only be of short-term therapeutic benefit and (2) expression of the corrective gene likely exerts its effect in the differentiated progeny, which may be numerous cell divisions downstream of the genetically modified stem cell. Transgene persistence can be accomplished, in principle, either by integration into one of the existing chromosomes or by incorporation of the transgene in a synthetic human microchromosome.

Viral Vectors Over the past two decades or more, significant attention has been devoted to the development of viral vectors and transduction protocols capable of stable introduction of genetic information into stem cells. Retrovirus, lentivirus, and adeno-associated virus (AAV) vectors are the most common vectors used in transduction of HSCs (Miller et al., 1990; Larochelle et al., 1996; Sutton et al., 1998; Uchida et al., 1998; Guenechea et al., 2000; May et al., 2000; Bradfute and Goodell, 2003) (reviewed in Hawley, 2001 and Baum et al., 2003). Applications using foamy virus suggest that these vector systems are also capable of marking engrafting stem cells as assayed in NOD/SCID mice (Hirata et al., 1996; Zucali et al., 2002). After several years of disappointing results, reports in humans (Aiuti et al., 2002) and other primates (Horn et al., 2002a,b), most particularly the French report of successful treatment of X-linked severe combined immune deficiency (SCID) (Cavazzana-Calvo et al., 2000), indicated that viral approaches can be successful in treating specific HSC-based diseases; however, a number of potential difficulties must be overcome to make this a safe and effective approach for stem cell gene therapy (discussed later).

Nonviral Integration Strategies Other approaches have been developed that allow for integration into the genome without use of viral vectors. Stable

939

integration of large DNA sequences into specific “attP sites” in the genome can be accomplished using integrase from bacteriophage phiC31 (Sclimenti et al., 2001; Olivares et al., 2002; Ortiz-Urda et al., 2002). This method for introducing large DNA, including needed regulatory elements, may allow genetic correction of inherited diseases caused by mutations in large genes. As well, transposon-based systems have been designed to allow for the insertion of transgenes into the human genome without the use of integrating viral vectors. Indeed, studies with human somatic stem cells in vitro and murine models in vivo indicate that Sleeping Beauty transposable elements may be able to correct certain genetic diseases (Montini et al., 2002; Ortiz-Urda et al., 2003). However, optimal transposon size may prevent the inclusion of larger genes or necessary regulatory elements (Ortiz-Urda et al., 2003).

Synthetic Microchromosomes Synthetic microchromosomes (SMCs), containing centromeric and telomeric sequences derived from functional human chromosomes, demonstrate significantly increased persistence and regulation of copy number in comparison with episomal constructs based on EpsteineBarr virus. SMCs have been shown to be mitotically and cytogenetically stable and segregated appropriately in a cycling human tumor cell line (Harrington et al., 1997). SMCs accommodate insertion of genetic regions that are sufficient to house therapeutic transgenes together with crucial intron/exon structure and regulatory sequences conferring appropriate transgene regulation. Whether transgene sequences maintained on SMCs are subject to the dysregulated expression and silencing that affect integrated sequences remains to be determined. Crucial issues with application of these SMCs to stem cell gene therapy will be the efficient delivery of single-copy SMCs to target cells and persistence of these SMCs during cell division and differentiation.

GENOME EDITING The second approach, genome editing, uses DNA repair and/or homologous recombination processes to correct an existing defective gene sequence so that the defective or mutated area of the gene is restored to a corrected normal state. Genome editing (as shown in Figure 77.1) involves the delivery of small DNA fragments, hybrid DNA/RNA molecules, and/or modified DNA polymers that are homologous to the target gene sequence with the exception of the base or bases intended for alteration. The genome editing process is directed by endogenous cellular machinery (potentially including mismatch repair and homologous recombination) acting at these target bases and

940

the sequence mismatches created. The target bases are exchanged for the bases present on the introduced DNA fragment e correcting or repairing the gene. The genetic alterations exacted are specific, targeted, and permanent. Repairing the defective sequence itself maintains the corrected genetic material within its normal chromatin environment, ensuring appropriate genetic regulation and expression in the cell. Genetic diseases resulting from welldefined, limited alterations in the DNA sequence, such as sickle cell disease, are ideal candidates for genome-editingbased gene therapy strategies. As well, genome editing may be the only suitable strategy in situations in which mutant gene product exercises a dominant negative influence over the normal gene product. For example, overexpression of normal collagen cannot surmount the harmful effects of mutant collagen chains produced in the disease osteogenesis imperfecta (Gajko-Galicka, 2002). Several classes of genome-editing molecules display conversion frequencies significantly higher than traditional gene-targeting methods in mammalian cells and have potential for clinical benefit (Cole-Strauss et al., 1996; Kunzelmann et al., 1996; Xiang et al., 1997; Goncz et al., 1998; Bandyopadhyay et al., 1999; Kren et al., 1999; Goncz and Gruenert, 2000). As well, recent advances in AAV vector development suggest these constructs may be capable of gene repair at frequencies sufficient for therapy (Russell et al., 2002). Recently, a report suggested that increased homologous recombination and gene correction can be obtained by combining triplex homing molecules and DNA or RNAeDNA hybrids e a technology called guided homologous recombination (GOREC) (Maurisse et al., 2002).

RNAeDNA Hybrids

VOLUME | 2

Adult and fetal Stem Cells

Brachman and Kmiec, 2003). Studies in yeast suggest that these molecules act by creating a single mismatched base pair on hybridization with the complementary sequence in the chromosome, which is then recognized and corrected by endogenous DNA repair machinery (Brachman and Kmiec, 2003). Successful application of these molecules to genome editing in mammalian cells has been reported (Liu et al., 2003).

Small Fragment Homologous Replacement (SFHR) SFHR uses short (typically 50 to 800 nucleotides) single- or double-stranded DNA fragments (SDFs) to alter one or more nucleotides of a specific sequence in the chromosome of a living cell (Kunzelmann et al., 1996; Goncz et al., 1998; Goncz and Gruenert, 2000; Goncz et al., 2001, 2002). The SDFs typically span the exon targeted for correction, with the terminal DNA sequences extending into non-exon regions. The precise cellular mechanism(s) responsible for SFHR-mediated genome editing remains to be elucidated. SDFs have been shown to efficiently (~0.1e10%) correct or modify specific sequences in genes known to be responsible for disease in several human and mouse transformed and primary cell lines (Kunzelmann et al., 1996; Goncz et al., 1998, 1999, 2001; Bruscia et al., 2002; Kapsa et al., 2002). Recent reports describing the targeting of the CFTR gene in mouse lung epithelial cells and the b-globin gene in human hematopoietic stem/progenitor cells (HSPCs) suggest therapeutic potential for this technology (Davis et al., 2002; Goncz et al., 2002). Furthermore, studies have demonstrated that SFHR-mediated genome editing can exact the modification of up to five bases within a given region (Colosimo et al., 2000).

These molecules, containing a central stretch of DNA bases (typically five bases) with the “correcting” sequence flanked by short stretches of RNA that form hairpin loops at the molecule ends, have demonstrated repair of both single base-pair substitutions and deletions. The gene correction mechanism is believed to involve mismatch repair (Cole-Strauss et al., 1996; Xiang et al., 1997; Liu et al., 2002). RNAeDNA hybrid molecules have been shown able to mediate correction of the sickle cell mutation in lymphoblastoid cell lines (Cole-Strauss et al., 1996) and introduce the sickle cell mutation into CD34þ cells from normal individuals (Xiang et al., 1997; Liu et al., 2002).

Triplex-Forming Oligonucleotides (TFOs)

Single-Stranded DNA Oligonucleotides

Although initial attempts to use appropriately constructed linear single-stranded AAV vectors in gene repair resulted in low levels of targeted gene repair, recent improvements have provided for targeted replacement of up to 1 kb into human chromosomes without additional mutation to the genome (Russell and Hirata, 1998; Inoue et al., 1999;

Modified synthetic single-stranded DNA oligonucleotides, approximately 25 to 74 bases in length, have also been found capable of single nucleotide exchange in a variety of organisms (Liu et al., 2001; Parekh-Olmedo et al., 2001;

Triplex DNA can be used to introduce mutations or genetic modifications in certain gene sequences (Wang et al., 1996). Triple helices, formed when the TFOs bind in the major groove of duplex DNA at polypurinee polypyrimidine sequences, have the ability to induce mutations in mammalian cells. A major limitation of this technology is the requirement for specific GC-rich repeats for sequence recognition.

Adeno-associated Virus Vectors

Chapter | 77

Stem Cell Gene Therapy

Hirata et al., 2002; Russell et al., 2002). AAV vectors have also demonstrated efficient correction of single-base mutations in marker genes including HPRT and alkaline phosphatase (Inoue et al., 2001). The random integration of AAV vectors into chromosomal DNA represents a potential drawback to this approach. Addition of accessory molecules may also provide for increased genome editing rates. For example, up to 100fold increases in gene targeting mediated by AAV have been observed following the introduction of DNA doublestrand breaks with sequence-specific nuclease (Miller et al., 2003; Porteus et al., 2003). Indeed, induced DNA doublestrand breaks using sequence-specific nucleases have also been shown effective in increasing the efficiency of nonAAV targeted gene repair in human cells (Porteus and Baltimore, 2003). Much debate surrounds the reported efficiencies of genome editing by many of the previously listed molecules. For example, it is well recognized that the residual presence of significant quantities of genome-editing molecules for several days posttransfection can result in polymerase chain reaction (PCR) artifacts, giving false evidence for conversion. Indeed, application of these technologies to stem cell gene therapy will require definitive proof of their merit in genome editing of chromosomal targets. Required demonstration of efficacy includes molecular evidence for genome editing at later times posttransfection, phenotypic demonstration of genome editing, Southern blot confirmation of the genome editing event, and demonstration of the absence of nonspecific gene conversion and absence of random integration of genome editing molecules in target cells. Such evidence will allow for further application of these powerful tools.

REQUIREMENTS FOR SUCCESSFUL STEM CELL GENE THERAPY Genetic Modification Directly in Stem Cells For long-term therapeutic benefit, it will typically be insufficient to modify only progenitor or differentiated cells; instead, direct genetic modification in stem cells will be required for sustained availability and production of corrected progeny cells. It is also essential that this genetic modification be accomplished without loss of stem cell activity.

Gene Addition Since the mid-1980s, retrovirus vectors have been the vehicles of choice for delivering transgenes (typically cDNAs together with limited transcriptional regulatory sequence) to cells, because they facilitate efficient

941

transgene delivery and integration into the chromosomal DNA of proliferating target cells. For example, efficient transduction of MAPCs and MSCs with standard retroviral vectors has been reported (Lou et al., 1999; Evans et al., 2000; Baxter et al., 2002; Jiang et al., 2002; Reyes et al., 2002). The particular challenge of transducing quiescent HSCs was a primary impetus for significant improvements in transduction technology, optimization of in vitro transduction conditions including choice of cytokines and use of retronectin, use of various viral envelopes, and development of lentivirus vectors and AAV vectors (although there is controversy regarding the AAV integration efficiency in HSCs (Srivasta, 2002)). The limited packaging size of retroviral, lentiviral, and AAV vectors precludes packaging of certain cDNAs (e.g., the complete dystrophin cDNA [14-kb cDNA] is too large to be incorporated into the previously mentioned vectors). The packaging of cDNAs means that only one gene product will typically be expressed, as opposed to the possible expression of alternatively spliced forms from the normal genomic configuration. These transduction methods allow for delivery without loss of stem cell activity. However, questions still remain as to the ability of these constructs to maintain long-term expression in the stem cells and their differentiated progeny, especially because the limited packing size of the vectors often precludes inclusion of key regulatory elements, exon/intron structure, and necessary insulator sequences (see the section on physiologically appropriate expression levels of the corrected gene product in the relevant cells).

Genome Editing Stem cell genome editing requires delivery of genomeediting molecules to stem cells without loss of stem cell function and successful editing of genomic sequences directly in the stem cells. Ideally, one would want efficient, quantitative delivery of genomic editing molecules to the nuclei of stem cells. Typical nonviral macromolecule delivery methodologies face inherent and/or potential limitations in human stem cells. For example, electroporation or liposome-mediated transfection conditions have not yet been reported that allow for the efficient delivery of macromolecules to HSCs without significant loss of viability or stem cell function (Toneguzzo and Keating, 1986; Harrison et al., 1995). In addition, these methods typically give rise to cells having significant cellto-cell variation in the number of DNA molecules transfected. The microinjection technology described in the section on genome editing of human HSPCs was developed for delivery of macromolecules to both human HSCs and MSCs in an effort to alleviate the problems inherent to traditional nonviral methods. It is not presently known whether the efficiency of editing chromosomal sequences is affected by the cycling

942

status of stem cells. Although not an issue for those stem cells that can apparently be maintained in a proliferative state in vitro without loss of stem cell function (e.g., MAPCs and MSCs), the question of whether replication of the target gene chromosomal DNA is required for successful genome editing is important for quiescent HSCs. It is perhaps important to note that the various genomeediting molecules summarized previously may use different mechanisms for genome editing and may, therefore, exhibit different requirements for cycling. It also remains to be determined whether efficient genome editing requires active transcription and/or an open chromatin conformation of the target gene. Although some genes requiring modification will already be expressed in stem cells (e.g., housekeeping genes), there are other target genes that would normally not be expressed until a certain state of differentiation or activation has been reached. Although it is assumed that stem cells in a quiescent state normally perform ongoing surveillance and repair of chromosomal mutations in both transcriptionally active and inactive genes, to our knowledge this has not been directly examined.

Genetically Corrected Stem Cells and Their Relevant Differentiated Progeny Consistently Present at Sufficient Frequency In Vivo A critical issue in stem cell gene therapy is achieving and maintaining a level of corrected stem cells and/or their progeny in vivo sufficient to achieve the desired therapeutic effect. Transplanted genetically modified stem cells and their progeny will exist in vivo in a background of transplanted unmodified cells and endogenous, unmodified stem cells and their progeny. The percentage of “corrected” cells required for therapeutic value will differ for various diseases. Factors that will influence the percentage of genetically modified cells in vivo are: 1. the percentage and number of genetically modified stem cells ex vivo before transplantation; 2. the ability to expand the stem cells ex vivo; 3. the ability to selectively expand or select for the genetically modified stem cells ex vivo or in vivo (at the expense of the endogenous, defective stem cells); and 4. whether the genetically modified cells (either stem cells or their differentiated progeny) have a proliferative, survival, or functional advantage in vivo. If there was a capability for significant ex vivo expansion of stem cells, without loss of stem cell biologic activity (including self-renewal and differentiation capability), the number of genetically modified stem cells delivered to the patient could be significantly increased, potentially

VOLUME | 2

Adult and fetal Stem Cells

increasing the frequency of genetically corrected versus endogenous defective stem cells in vivo following transplant. In addition, if there existed the ability to obtain, ex vivo, expanded populations of cells all derived from a single stem cell clone, cells for transplantation could be prepared that were precharacterized for their site of retroviral integration (to eliminate clones with problematic insertional mutagenesis sites or to identify integrants likely to demonstrate appropriately regulated transgene expression) or for successful genome editing (i.e., only delivering appropriately corrected stem cells). Studies performed with human MSCs or human MAPCs suggest that these classes of stem cells may be capable of significant ex vivo expansion (Bruder et al., 1997; Pittenger et al., 1999; Jiang et al., 2002; Reyes et al., 2002; Qi et al., 2003).

Gene Addition Inclusion of a selectable marker gene, in addition to the therapeutic transgene, in the same viral vector will, in principle, permit ex vivo or in vivo selection of genetically modified stem cells and their progeny. For example, various groups have examined the ability to specifically select in vivo for HSCs and progeny transduced with a retroviral vector expressing either normal O6 methylguanine methyltransferase (MGMT) that confers resistance to nitrosoureas, such as BCNU, or a mutant MGMT (e.g., P140K) resistant to the combination of O6-benzylguanine plus BCNU (Allay et al., 1996; Davis et al., 1997; Koc et al., 1999; Davis et al., 2000; Lee et al., 2001; Zielske and Gerson, 2002). Other selectable genes have included dihydroxyfolate reductase (DFHR) and multiple drug resistance (MDR) (Sellers et al., 2001). Although these studies have demonstrated excellent in vivo selection for the genetically modified cell, a concern remains with regard to potential in vivo toxicity at time of chemotherapeutic treatment or incidence of cancer in later years because of the chemotherapeutic treatment(s).

Genome Editing Genome editing will most likely first find application when there is a natural in vivo selective advantage for genetically corrected stem cells and/or their progeny, e.g., Wiskott Aldrich syndrome (WAS), Fanconi’s anemia, or X-SCID. There should be a strong selective advantage in vivo for the corrected HSCs and their T-lymphocyte progeny in WAS as evidenced by studies of X-chromosome inactivation patterns in female WAS carriers (Greer et al., 1989; Mantuano et al., 1993; Wengler et al., 1995) and the selective reversion to a corrected WASP allele in a patient with WAS (Wada et al., 2001). These data strongly suggest that even the correction of a small number of HSCs with the capability of differentiating into gene-corrected T-cell progenitors and mature T lymphocytes may lead to

Chapter | 77

Stem Cell Gene Therapy

significant clinical benefit in WAS. This suggestion is supported by recent efforts to treat X-SCID via stem cell gene therapy (Wengler et al., 1993; Stephan et al., 1996). It is believed that strong in vivo selective pressure for T and B cells expressing the gamma chain of the interleukin (IL)-2 receptor contributed to the success of theX-SCID trial in France (Cavazzana-Calvo, 2000). For those diseases in which the genome-edited stem cells and/or their progeny do not have a selective advantage in vivo, successfully genome-edited stem cells could be subsequently transduced with constructs having selectable genes (e.g., P140K MGMT) as described previously. Use of systems such as Cre-Loc could facilitate excision of drug selection constructs following in vivo selection.

Physiologically Appropriate Expression Levels of the Corrected Gene Product in the Relevant Cells Correction of genetic deficiencies will typically require that the therapeutic gene be expressed at the appropriate level in the relevant cells for the life of the patient. Genetic treatment of some diseases will have less stringent requirements for either the level of required expression (e.g., chronic granulomatous disease) or the ability to tolerate indiscriminate expression (e.g., adenosine deaminase deficiency, Gaucher disease). In contrast, successful treatment of other diseases (e.g., hemoglobinopathies, X-linked agammaglobulinemia) will likely require expression that is both of sufficient level and cell-type specific (Persons and Nienhuis, 2000). For example, it is expected that clinical benefit in hemoglobinopathies will require that nonerythroid cells remain absent of globin expression and that most erythrocytes will express the inserted gene at greater than 10e20% of normal globin levels for thalassemia and 20e40% of HbF levels for sickle cell disease (McInerney et al., 2000). Finally, ectopic constitutive expression of therapeutic transgenes may be harmful in cases in which expression of the endogenous gene is tightly regulated (e.g., with respect to the cell’s activation state). For example, although ectopic expression of the CD40L transgene corrected a CD40L deficiency in mice it also caused a thymic lymphoproliferative disease (Brown et al., 1998).

Gene Addition Significant problems have been encountered in satisfying the requirement for long-term, cell-type-specific transgene expression. For example, the expression of retrovirustransduced transgenes is frequently silenced in the progeny of transduced human or primate progenitors (Challita and Kohn, 1994; Lu et al., 1994; Persons and Nienhuis, 2000). Furthermore, even in those cells demonstrating some

943

expression, there may be significant variability in the level of expression from cell to cell. The difficulty in achieving appropriate expression (proper level, regulation, and celltype specificity) is due to integration position effects, uneven distribution of the number of integrated gene copies per cell, and the inability to package sufficient regulatory sequences within the viral vector. As a consequence of this dysregulated expression, cells, although genetically modified with corrective genes, may not efficiently display the corrected phenotype. The variation in level of transgene expression from cell to cell is determined, to a large extent, by the site of proviral integration. For retroviral and lentiviral vectors, the site of integration is essentially random, although there is a preference for active or “bent” chromatin. Several recent studies have reported that multiple retroviral or lentiviral integration events per cell were required for adequate levels of transgene expression. For example, Woods et al. (2003) reported an average of 5.6  3.3 integration events per cell under efficient transduction conditions, and several groups have reported that multiple proviral integrations per cell were required for consistent therapeutic expression, presumably because some of the integration events are “silent” or expressed at only very low level (Kalberer et al., 2000; Pawliuk et al., 2001; Imren et al., 2002; Persons et al., 2003). The requirement for multiple integration events creates at least two problems: (1) increased likelihood of insertion mutagenesis (see later) and (2) a potential for inappropriately high expression of transgene in the cells (e.g., an overexpression of b globin may be manifest as a-thalassemia). Interestingly, these same features of dysregulated transgene expression were also observed in the early transgenic mouse expression studies (Pasceri et al., 1998). Subsequent studies demonstrated that long-term, positionindependent, copy-number-dependent, cell-type-specific expression required strong promoter/enhancer elements, sufficient genomic sequences to dominantly confer the appropriate chromatin configuration e i.e., an open chromatin conformation in expressing cells, e.g., inclusion of locus control region (LCR)-like elements (Einerhand et al., 1995; Sadelain et al., 1995; Pasceri et al., 1998) e and sufficient intron/exon structure and sequences for highlevel expression (Persons and Nienhuis, 2000). The strict packaging requirements for retrovirus, lentivirus, and AAV vectors may preclude inclusion of sufficient regulatory sequences and/or intron/exon structure for therapeutic applications requiring highly regulated gene expression. Strong splicing signals (i.e., intron/exon structure) or cryptic splicing signals in transgene sequences (e.g., when inserted in reverse orientation) may interfere with packaging of the desired unspliced full-length construct (Leboulch et al., 1994). Certain position effects may be overcome via use of insulator sequences; whether their

944

inclusion will suffice to ensure accurate and regulated expression is under active investigation (Emery et al., 2000, 2002). The complex and coordinate regulation of globin gene expression in vivo raises significant challenges for treating sickle cell anemia or b-thalassemia by addition of a normal or specially designed globin gene using retroviral, lentiviral, or AAV vectors. Much effort has been dedicated to identifying and including in viral vectors the regulatory sequences that are necessary and sufficient for appropriately regulated transgene expression. Recently, use of optimized vector constructs have yielded significant improvements in long-term, erythroid-specific globin expression in mouse models (Kalberer et al., 2000; May et al., 2000, 2002; Pawliuk et al., 2001; Emery et al., 2002; Imren et al., 2002; Nicolini et al., 2002; Persons et al., 2003a,b; Rivella et al., 2003). Whether these very promising results will be confirmed in human clinical trials remains to be determined. A significant challenge for achieving appropriately regulated transgene expression via stem cell gene therapy is that the transgene, together with regulatory sequences, is delivered into a chromatin environment (specifically, stem cell) that may be significantly different from the chromatin environment in which the transgene expression is ultimately required (e.g., erythroid for globin expression; T-cell for WASP expression). For example, a transgene may integrate into an active chromatin locus in stem cells that may subsequently become an inactive chromatin locus in the differentiated cell type(s). Furthermore, it is not known whether transcriptional regulatory sequences directly incorporated into stem cell chromatin will show the same loading of chromatin remodeling and transcription factors as when the transcriptional regulatory sequences are present in the chromatin from the stage of embryonic stem cell onward. In other words, is chromatin structure (including histones, transcription factors, etc.) a consequence of sequential steps of factor addition and removal, originating in the fertilized egg or embryonal stem cell? Or can it be created de novo from factors present in the HSC? For example, Vassilopoulos et al. (1999) reported that appropriate activity of the b-globin regulatory sequences required passage through a nonerythroid environment.

Genome Editing Repairing the mutation itself within the defective gene (e.g., b-globin gene) would maintain the correct genetic material within its normal chromatin environment and in principle ensure appropriate genetic regulation and expression in the progeny differentiated cells (e.g., erythroid cells). This is one of the primary advantages of the genome editing approach for genes in which the transcriptional regulatory sequences are intact.

VOLUME | 2

Adult and fetal Stem Cells

Absence of Interference from Endogenous Defective Gene Product on the Activity of the Corrected Gene Product For those situations in which there is ongoing expression of the endogenous defective gene, a critical issue is whether this mutant gene product will interfere with the function of the introduced correct transgene. For example, it is possible that endogenous expression of a truncated or mutant protein could interfere with the functioning of an introduced normal protein in a dominant negative manner (e.g., collagen or WASP). Persistence of the mutant protein may be a particular problem in cases in which the gene product normally forms homodimers or heterodimers or trimers (e.g., CD40L).

Gene Addition Addition of a normal gene into a stem cell means that the defective gene is still present within that cell and capable of action. If ongoing expression of the endogenous defective gene is detrimental to the cell, it may be necessary to either express a specific transgene product that is designed to specifically counteract the activity of the mutant gene (e.g., expression of an anti-sickling gene) or to overexpress the normal transgene product at a level sufficient to dilute out the effect of the mutant gene product. These approaches may not be possible in all cases because overexpression of the normal gene product may itself generate negative side effects or unexpected results.

Genome Editing Genome editing may be the only suitable genetic modification in situations in which the mutant gene product exercises a dominant negative influence over the normal gene product, because this approach would convert the endogenous gene to a normal form, ablating the detrimental mutant gene.

Absence of Adverse Effects Of critical importance in any therapy is minimization of side effects or adverse effects caused by the treatment itself. A partial listing of potential adverse effects from stem cell gene therapy would include insertional mutagenesis from transgene integration, nonspecific gene editing introducing untoward mutations, and inadvertent carcinogenesis from the in vivo selection method for corrected cells. Although a complete absence of any adverse effects is clearly the goal of any genetic therapy, the potential risks of any treatment must be assessed in concert with the potential benefits. For some of the diseases potentially amenable to stem cell gene therapy, the current prognosis is very poor with no other therapy available. It should also be remembered that the

Chapter | 77

Stem Cell Gene Therapy

introduction of other groundbreaking therapies (e.g., heart transplantation, marrow transplantation) resulted in numerous initial failures, including death of patients. In addition, even today, therapies such as allogenic marrow transplantation for primary immunodeficiency disease are only 90% successful.

Gene Addition A difficulty of the gene addition approach is the potential leukemogenicity resulting from random integration of the introduced viral vector into chromosomal DNA (e.g., either activating oncogenes or disabling tumor suppressor genes). This possibility has been reported both in retroviral transduction of mouse stem cells (Li et al., 2002) and in the otherwise successfulX-SCID human trial in France (Cavazzana-Calvo et al., 2000). Furthermore, Woods et al. (2003) reported that efficient lentiviral vector transduction of human NOD-SCID repopulating cells yielded multiple integrations per cell e increasing the chance of insertional mutagenesis. In fact, one of the characterized integrants was localized in the BRCA1 tumor suppressor gene.

Genome Editing One of the crucial criteria for genome editing is the specificity of the editing event. A balance between specific and nonspecific genome editing may occur and as such it is imperative to develop methods that favor specific actions. As well, use of methods that introduce double-strand DNA breaks (believed to increase the rates of repair) requires strict regulation to ensure that these breaks are limited and specific to the target sequence (e.g., through engineering of site-specific nucleases). Some of the molecules used in genome editing also have the potential for random integration within the genome and, therefore, possess the potential to disrupt genes and/or cause mutagenesis. Assessment of this risk or application of molecules that do not randomly integrate into the genome will be necessary for therapeutic application.

GENOME EDITING OF HUMAN HEMATOPOIETIC STEM/PROGENITOR CELLS One of the current limitations to development of genomeediting strategies in human adult stem cells, and particularly HSPCs, has been the difficulty in achieving efficient delivery of genome-editing molecules to the nucleus of stem cells without loss of stem cell activity. This section summarizes our development of a novel method for delivering macromolecules to the nuclei of HSPCs and MSCs and our experience, in particular, with genome editing of HSPCs.

945

Microinjection-mediated Delivery of Macromolecules to Adult Stem Cells Although therapeutic application will likely require the correction of a large number of HSPCs (given current identification procedures for engrafting cells) and, therefore, a robust method for bulk delivery of macromolecules to the patient’s cells, microinjection provides an ideal experimental tool for quantitative delivery of macromolecules to the nuclei of HSPCs (Davis et al., 2000a,b). With the development of strategies for attachment of HSPCs to extracellular matrix-coated dishes without affecting cell function and the developmentof injection needles with very small outer tip diameters (OTDs) (~0.2 mm) that do not damage these relatively small cells (~6 mm diameter for stem cells (Berardi et al., 1985)), glass-needle-mediated microinjection technology has been successfully applied to HSPCs (Davis et al., 2000a,b). Importantly, macromolecule delivery is accomplished with high postinjection cell viability (up to 87% postinjection viabilities in CD34þ/ CD38e cells), no discernible impact on stem/progenitor cell proliferation or biologic activity, and a high frequency of cells expressing injected transgenes (Davis et al., 2000b). Microinjection can deliver macromolecules into cells irrespective of whether the cells are in a quiescent or cycling state, and by regulating concentration of DNA injected, flow rate, and injection time per cell, the number of molecules delivered to a cell can be approximately controlled. Microinjection has been demonstrated to be a well-tolerated method for the delivery of various genome editing molecules to HSPCs (Goncz et al., 2002; Liu et al., 2002). These microinjection technologies have been expanded to MSCs with postinjection viability and shortterm GFP gene expression greater than 60% (Tsulaia et al., 2003).

Genome Editing of the b-globin Gene in Hematopoietic Stem/Progenitor Cells Genome editing strategies have been used to introduce the sickle cell disease lesion, a single base-pair transversion (A / T), in codon 6 of the b-globin gene in normal HSPCs. Targeted modification of the b-globin gene has been assessed following delivery via microinjection of both RNAeDNA hybrids (Liu et al., 2002) and SFHR molecules (Davis et al., 2002; Gonz et al., 2002).

RNAeDNA Hybrids Liu et al. (2002) described the application of RNAeDNA hybrids to site-specific nucleotide exchange in the human globin gene in primitive human blood cells. RNAeDNA hybrids delivered via microinjection to CD34þ and LineCD38e cells resulted in the A to T nucleotide

946

exchange in 23% of experiments analyzed. Furthermore, conversion of the b-globin gene was detected in the erythroid progeny of LineCD38e cells at the mRNA level. Interestingly, conversion rates as high as 10e15% were seen in some experimental samples, suggesting that levels of conversion may be sufficient to achieve therapeutic benefit in patients with sickle cell disease.

Small Fragment Homologous Replacement The feasibility of using SFHR in the modification of normal human b (bA)-globin sequences was assessed in engrafting normal HSPCs. Short DNA fragments (bS SDF, 559 bp) made up of sickle b (bS)-globin sequence were microinjected into the nuclei of Line/CD38e cells. Site-specific conversion (bA / bS-globin) was observed in 42% of the experiments (70 experiments total) as determined by DNA and RNA analysis at 2 to 7 weeks postinjection. The percent fraction of b-globin alleles that were converted in each experiment ranged between 1% and 13% (Davis et al., 2002; Goncz et al., 2002). Line/CD38e cells, microinjected with bS SDF, were also transplanted into irradiated NOD/SCID/b2 microglobulin knockout mice to assess whether bA to bS conversion had actually occurred in cells capable of engrafting the bone marrow of these mice. Successful engraftment of genetically modified primitive human blood cells in immunedeficient mice was observed with significant conversion of the b-globin gene (bA to bS) (Davis et al., 2002; Goncz et al., 2002). Evidence for the presence of sickle globin protein in modified cells and/or demonstration of genome editing of HSPCs isolated from sickle cell patients will provide further support for application of genome editing technologies in ex vivo stem cell gene therapy.

CONCLUSION Gene addition and genome-editing approaches as applied to human stem cells offer significant therapeutic potential. It is most likely that clinical benefit will be initially demonstrated in diseases in which there is a clear selective advantage (e.g., proliferative, functional, or survival) for the corrected stem cells or their progeny and/or in which the requirements for regulation of gene expression are less stringent. Significant improvements in both technologies will be required to bring about therapeutic benefit to a wider range of inherited genetic diseases.

REFERENCES Aiuti, A., Slavin, S., Aker, M., Ficara, F., Deola, S., Mortellaro, A., et al., 2002. Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science 296, 2410e2413.

VOLUME | 2

Adult and fetal Stem Cells

Allay, J.A., Koc, O.N., Davis, B.M., Gerson, S.L., 1996. Retroviralmediated gene transduction of human alkyltransferase complementary DNA confers nitrosourea resistance to human hematopoietic progenitors. Clin. Cancer Res. 2, 1353e1359. Bandyopadhyay, P., Ma, X., Linehan-Stieers, C., Kren, B.T., Steer, C.J., 1999. Nucleotide exchange in genomic DNA of rat hepatocytes using RNA/DNA oligonucleotides: targeted delivery of liposomes and polyethylenimine to the asialoglycoprotein receptor. J. Biol. Chem. 274, 10163e10172. Baum, C., Dullmann, J., Li, Z., Fehse, B., Meyer, J., Williams, D.A., et al., 2003. Side effects of retroviral gene transfer into hematopoietic stem cells. Blood 101, 2099e2114. Baxter, M.A., Wynn, R.F., Deakin, J.A., Bellantuono, I., Edington, K.G., Cooper, A., et al., 2002. Retrovirally mediated correction of bone marrow-derived mesenchymal stem cells from patients with mucopolysaccharidosis type I. Blood 99, 1857e1859. Berardi, A.C., Wang, A., Levine, J.D., Lopez, P., Scadden, D.T., 1995. Functional isolation and characterization of human hematopoietic stem cells. Science 267, 104e108. Brachman, E.E., Kmiec, E.B., 2003. Targeted nucleotide repair of cyc1 mutations in Saccharomyces cerevisiae directed by modified singlestranded DNA oligonucleotides. Genetics 163, 527e538. Bradfute, S.B., Goodell, M.A., 2003. Adenoviral transduction of mouse hematopoietic stem cells. Mol. Ther. 7, 334e340. Brown, M.P., Topham, D.J., Sangster, M.Y., Brown, M.P., Topham, D.J., Sangster, M.Y., et al., 1998. Tymic lymphoproliferative disease after successful correction of CD40 ligand deficiency by gene transfer in mice. Nat. Med. 4, 1253e1260. Bruder, S.P., Jaiswal, N., Haynesworth, S.E., 1997. Growth kinetics, selfrenewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J. Cell. Biochem. 64, 278e294. Bruscia, E., Sangiuolo, F., Sinibaldi, P., Goncz, K.K., Novelli, G., Gruenert, D.C., 2002. Isolation of CF cell lines corrected at DeltaF508-CFTR locus by SFHR-mediated targeting. Gene. Ther. 9, 683e685. Cavazzana-Calvo, M., Hacein-Bey, S., de Saint, B.G., Gross, F., Yvon, E., Nusbaum, P., et al., 2000. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 288, 669e672. Challita, P.M., Kohn, D.B., 1994. Lack of expression from a retroviral vector after transduction of murine hematopoietic stem cells is associated with methylation in vivo. Proc. Natl. Acad. Sci. U.S.A. 91, 2567e2571. Cole-Strauss, A., Yoon, K., Xiang, Y., Byrne, B.C., Rice, M.C., Gryn, J., et al., 1996. Correction of the mutation responsible for sickle cell anemia by an RNA- DNA oligonucleotide. Science 273, 1386e1389. Colosimo, A., Goncz, K.K., Holmes, A.R., Kunzelmann, K., Novelli, G., Malone, R.W., et al., 2000. Transfer and expression of foreign genes in mammalian cells. Biotechniques. 29, 314e318. 320e322, 324. Davis, B.M., Koc, O.N., Gerson, S.L., 2000. Limiting numbers of G156A O(6)-methylguanine-DNA methyltransferase-transduced marrow progenitors repopulate nonmyeloablated mice after drug selection. Blood 95, 3078e3084. Davis, B.M., Reese, J.S., Koc, O.N., Lee, K., Schupp, J.E., Gerson, S.L., 1997. Selection for G156A O6-methylguanine DNA methyltransferase gene-transduced hematopoietic progenitors and protection from lethality in mice treated with O6-benzylguanineand 1,3-bis(2-chloroethyl)-1-nitrosourea. Cancer Res. 57, 5093e5099.

Chapter | 77

Stem Cell Gene Therapy

Davis, B.R., Brown, D.B., Prokopishyn, N.L., Yannariello-Brown, J., 2000a. Micro-injection-mediated hematopoietic stem cell gene therapy. Curr. Opin. Mol. Ther. 2, 412e419. Davis, B.R., Yannariello-Brown, J., Prokopishyn, N.L., Luo, Z., Smith, M.R., Wang, J., et al., 2000b. Glass needle-mediated microinjection of macromolecules and transgenes into primary human blood stem/progenitor cells. Blood 95, 437e444. Davis, B.R., Prokopishyn, N.L., Goncz, K.K., Gruenert, D.C., 2002. Sequence specific genomic editing of the beta-globin gene in human blood stem/progenitor cells. Blood 100, 183a. Einerhand, M.P., Antoniou, M., Zolotukhin, S., Muzyczka, N., Berns, K.I., Grosveld, F., et al., 1995. Regulated high-level human beta-globin gene expression in erythroid cells following recombinant adenoassociated virus-mediated gene transfer. Gene. Ther. 2, 336e343. Emery, D.W., Yannaki, E., Tubb, J., Stamatoyannopoulos, G., 2000. A chromatin insulator protects retrovirus vectors from chromosomal position effects. Proc. Natl. Acad. Sci. U.S.A. 97, 9150e9155. Emery, D.W., Yannaki, E., Tubb, J., Nishino, T., Li, Q., Stamatoyannopoulos, G., 2002. Development of virus vectors for gene therapy of beta chain hemoglobinopathies: flanking with a chromatin insulator reduces gamma-globin gene silencing in vivo. Blood 100, 2012e2019. Evans, C.H., Ghivizzani, S.C., Smith, P., Shuler, F.D., Mi, Z., Robbins, P.D., 2000. Using gene therapy to protect and restore cartilage. Clin. Orthop. 379 (Suppl), S214eS219. Gajko-Galicka, A., 2002. Mutations in type I collagen genes resulting in osteogenesis imperfecta in humans. Acta. Biochim. Pol. 49, 433e441. Goncz, K.K., Gruenert, D.C., 2000. Site-directed alteration of genomic DNA by small-fragment homologous replacement. Methods Mol. Biol. 133, 85e99. Goncz, K.K., Kunzelmann, K., Xu, Z., Gruenert, D.C., 1998. Targeted replacement of normal and mutant CFTR sequences in human airway epithelial cells using DNA fragments. Hum. Mol. Genet. 7, 1913e1919. Goncz, K.K., Feeney, L., Gruenert, D.C., 1999. Differential sensitivity of normal and cystic fibrosis airway epithelial cells to epinephrine. Br. J. Pharmacol. 128, 227e233. Goncz, K.K., Colosimo, A., Dallapiccola, B., Gagne, L., Hong, K., Novelli, G., et al., 2001. Expression of DeltaF508 CFTR in normal mouse lung after site-specific modification of CFTR sequences by SFHR. Gene. Ther. 8, 961e965. Goncz, K.K., Prokopishyn, N.L., Chow, B.L., Davis, B.R., Gruenert, D.C., 2002. Application of SFHR to gene therapy of monogenic disorders. Gene. Ther. 9, 691e694. Greer, W.L., Kwong, P.C., Peacocke, M., Ip, P., Rubin, L.A., Siminovitch, K.A., 1989. X-chromosome inactivation in the WiskottAldrich syndrome: a marker for detection of the carrier state and identification of cell lineages expressing the gene defect. Genomics 4, 60e67. Gronthos, S., Zannettino, A.C., Hay, S.J., Shi, S., Graves, S.E., Kortesidis, A., et al., 2003. Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow. J. Cell Sci. 116, 1827e1835. Guenechea, G., Gan, O.I., Inamitsu, T., Dorrell, C., Pereira, D.S., Kelly, M., et al., 2000. Transduction of human CD34þ. Mol. Ther. 1, 566e573. Harrington, J.J., Van Bokkelen, G., Mays, R.W., Gustashaw, K., Willard, H.F., 1997. Formation of de novo centromeres and

947

construction of first-generation human artificial microchromosomes. Nat. Genet. 15, 345e355. Harrison, G.S., Wang, Y., Tomczak, J., Hogan, C., Shpall, E.J., Curiel, T.J., et al., 1995. Optimization of gene transfer using cationic lipids in cell lines and primary human CD4þ and CD34þ hematopoietic cells. Biotechniques 19, 816e823. Hawley, R.G., 2001. Progress toward vector design for hematopoietic stem cell gene therapy. Curr. Gene. Ther. 1, 1e17. Hirata, R.K., Miller, A.D., Andrews, R.G., Russell, D.W., 1996. Transduction of hematopoietic cells by foamy virus vectors. Blood 88, 3654e3661. Hirata, R., Chamberlain, J., Dong, R., Russell, D.W., 2002. Targeted transgene insertion into human chromosomes by adeno-associated virus vectors. Nat. Biotechnol. 20, 735e738. Horn, P.A., Topp, M.S., Morris, J.C., Riddell, S.R., Kiem, H.P., 2002a. Highly efficient gene transfer into baboon marrow repopulating cells using GALV-pseudotype oncoretroviral vectors produced by human packaging cells. Blood 100, 3960e3967. Horn, P.A., Morris, J.C., Bukovsky, A.A., Andrews, R.G., Naldini, L., Kurre, P., et al., 2002b. Lentivirus-mediated gene transfer into hematopoietic repopulating cells in baboons. Gene Ther. 1464e1471. Gene. Ther. 9, 1464e1471. Imren, S., Payen, E., Westerman, K.A., Pawliuk, R., Fabry, M.E., Eaves, C.J., et al., 2002. Permanent and panerythroid correction of murine beta thalassemia by multiple lentiviral integration in hematopoietic stem cells. Proc. Natl. Acad. Sci. U.S.A. 99, 14380e14385. Inoue, N., Hirata, R.K., Russell, D.W., 1999. High-fidelity correction of mutations at multiple chromosomal positions by adeno-associated virus vectors. J. Virol. 73, 7376e7380. Inoue, N., Dong, R., Hirata, R.K., Russell, D.W., 2001. Introduction of single base substitutions at homologous chromosomal sequences by adeno-associated virus vectors. Mol. Ther. 3, 526e530. Jiang, Y., Vaessen, B., Lenvik, T., Blackstad, M., Reyes, M., Verfaillie, C.M., 2002. Multipotent progenitor cells can be isolated from postnatal murine bone marrow, muscle, and brain. Exp. Hematol. 30, 896e904. Johansson, C.B., Momma, S., Clarke, D.L., Risling, M., Lendahl, U., Frisen, J., 1999. Identification of a neural stem cell in the adult mammalian central nervous system. Cell 96, 25e34. Kalberer, C.P., Pawliuk, R., Imren, S., Bachelot, T., Takekoshi, K.J., Fabry, M., et al., 2000. Preselection of retrovirally transduced bone marrow avoids subsequent stem cell gene silencing and age-dependent extinction of expression of human beta-globin in engrafted mice. Proc. Natl. Acad. Sci. U.S.A. 97, 5022e5024. Kapsa, R.M., Quigley, A.F., Vadolas, J., Steeper, K., Ioannou, P.A., Byrne, E., et al., 2002. Targeted gene correction in the mdx mouse using short DNA fragments: towards application with bone marrowderived cells for autologous remodeling of dystrophic muscle. Gene. Ther. 9, 695e699. Koc, O.N., Reese, J.S., Szekely, E.M., Gerson, S.L., 1999. Human longterm culture initiating cells are sensitive to benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea and protected after mutant (G156A) methylguanine methyltransferase gene transfer. Cancer Gene. Ther. 6, 340e348. Kren, B.T., Parashar, B., Bandyopadhyay, P., Chowdhury, N.R., Chowdhury, J.R., Steer, C.J., 1999. Correction of the UDP-glucuronosyltransferase gene defect in the gunn rat model of Crigler-Najjar syndrome type I with a chimeric oligonucleotide. Proc. Natl. Acad. Sci. U.S.A. 96, 10349e10354.

948

Kunzelmann, K., Legendre, J.Y., Knoell, D.L., Escobar, L.C., Xu, Z., Gruenert, D.C., 1996. Gene targeting of CFTR DNA in CF epithelial cells. Gene. Ther. 3, 859e867. Larochelle, A., Vormoor, J., Hanenberg, H., Wang, J.C., Bhatia, M., Lapidot, T., et al., 1996. Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy. Nat. Med. 2, 1329e1337. Leboulch, P., Huang, G.M., Humphries, R.K., Oh, Y.H., Eaves, C.J., Tuan, D.Y., et al., 1994. Mutagenesis of retroviral vectors transducing human beta-globin gene and beta-globin locus control region derivatives results in stable transmission of an active transcriptional structure. EMBO J. 13, 3065e3076. Lee, K., Gerson, S.L., Maitra, B., Koc, O.N., 2001. G156A MGMTtransduced human mesenchymal stem cells can be selectively enriched by O6-benzylguanine and BCNU. J. Hematother. Stem Cell Res. 10, 691e701. Li, Z., Dullmann, J., Schiedlmeier, B., Schmidt, M., von Kalle, C., Meyer, J., et al., 2002. Murine leukemia induced by retroviral gene marking. Science 296, 497. Liu, H., Agarwal, S., Kmiec, E., Davis, B.R., 2002. Targeted beta-globin gene conversion in human hematopoietic CD34(þ) and Lin(-)CD38(-) cells. Gene. Ther. 9, 118e126. Liu, L., Rice, M.C., Kmiec, E.B., 2001. In vivo gene repair of point and frameshift mutations directed by chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides. Nucleic Acids Res. 29, 4238e4250. Liu, L., Brachman, E.E., Drury, M.D., Sonntag, J.M., Maquire, K.K., Kmiec, E.B., 2003. Regulation of gene repair directed by synthetic end-modified oligonucleotides. Mol. Ther. 7, S308. Lou, J., Xu, F., Merkel, K., Manske, P., 1999. Gene therapy: adenovirusmediated human bone morphogenetic protein-2 gene transfer induces mesenchymal progenitor cell proliferation and differentiation in vitro and bone formation in vivo. J. Orthop. Res. 17, 43e50. Lu, M., Maruyama, M., Zhang, N., Levine, F., Friedmann, T., Ho, A.D., 1994. High efficiency retroviral-mediated gene transduction into CD34þ cells purified from peripheral blood of breast cancer patients primed with chemotherapy and granulocyte-macrophage colonystimulating factor. Hum. Gene. Ther. 5, 203e208. Mantuano, E., Candotti, F., Giliani, S., Parolini, O., Lusardi, M., Zucchi, M., et al., 1993. Analysis of X-chromosome inactivation in bone marrow precursors from carriers of Wiskott-Aldrich syndrome andX-linked severe combined immunodeficiency: evidence that the Wiskott-Aldrich gene is expressed prior to granulocyte-macrophage colony-forming-unit. Immunodeficiency 4, 271e276. Maurisse, R., Feugeas, J.P., Biet, E., Kuzniak, I., Leboulch, P., Dutreix, M., et al., 2002. A new method (GOREC) for directed mutagenesis and gene repair by homologous recombination. Gene. Ther. 9, 703e707. May, C., Rivella, S., Callegari, J., Heller, G., Gaensler, K.M., Luzzatto, L., et al., 2000. Therapeutic haemoglobin synthesis in betathalassaemic mice expressing lentivirus-encoded human beta-globin. Nature 406, 82e86. May, C., Rivella, S., Chadburn, A., Sadelain, M., 2002. Successful treatment of murine beta-thalassemia intermedia by transfer of the human beta-globin gene. Blood 99, 1902e1908. McCune, S.L., Reilly, M.P., Chomo, M.J., Asakura, T., Townes, T.M., 1994. Recombinant human hemoglobins designed for gene therapy of sickle cell disease. Proc. Natl. Acad. Sci. U.S.A. 91, 9852e9856.

VOLUME | 2

Adult and fetal Stem Cells

McInerney, J.M., Nemeth, M.J., Lowrey, C.H., 2000. Erythropoiesis: review article e slow and steady wins the race? Progress in the development of vectors for gene therapy of beta-thalassemia and sickle cell disease. Hematology 4, 437e455. Miller, D.G., Adam, M.A., Miller, A.D., 1990. Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol. Cell Biol. 10, 4239e4242. Miller, D.G., Petek, L.M., Russell, D.W., 2003. Human gene targeting by adeno-associated virus vectors is enhanced by DNA double-strand breaks. Mol. Cell Biol. 23, 3550e3557. Montini, E., Held, P.K., Noll, M., Morcinek, N., Al-Dhalimy, M., Finegold, M., et al., 2002. In vivo correction of murine tyrosinemia type I by DNA-mediated transposition. Mol. Ther. 6, 759e769. Nicolini, F.E., Imren, S., Oh, I.H., Humphries, R.K., Leboulch, P., Fabry, M.E., et al., 2002. Expression of a human beta-globin transgene in erythroid cells derived from retrovirally transduced transplantable human fetal liver and cord blood cells. Blood 100, 1257e1264. Olivares, E.C., Hollis, R.P., Chalberg, T.W., Meuse, L., Kay, M.A., Calos, M.P., 2002. Site-specific genomic integration produces therapeutic Factor IX levels in mice. Nat. Biotechnol. 20, 1124e1128. Ortiz-Urda, S., Thyagarajan, B., Keene, D.R., Lin, Q., Fang, M., Calos, M.P., et al., 2002. Stable nonviral genetic correction of inherited human skin disease. Nat. Med. 8, 1166e1170. Ortiz-Urda, S., Lin, Q., Yant, S.R., Keene, D., Kay, M.A., Khavari, P.A., 2003. Sustainable correction of junctional epidermolysis bullosa via transposon-mediated non-viral gene transfer. Gene. Ther. 10, 1099e1104. Parekh-Olmedo, H., Czymmek, K., Kmiec, E.B., 2001. Targeted gene repair in mammalian cells using chimeric RNA/DNA oligonucleotides and modified single-stranded vectors. Sci. STKE 2001 Mar 13; 2001(73):PL1. Pasceri, P., Pannell, D., Wu, X., Ellis, J., 1998. Full activity from human beta-globin locus control region transgenes requires 50 HS1, distal beta-globin promoter, and 30 beta-globin. Blood 92, 653e663. Pawliuk, R., Westerman, K.A., Fabry, M.E., Payen, E., Tighe, R., Bouhassira, E.E., et al., 2001. Correction of sickle cell disease in transgenic mouse models by gene therapy. Science 294, 2368e2371. Persons, D.A., Nienhuis, A.W., 2000. Gene therapy for the hemoglobin disorders: Past, present, and future. Proc. Natl. Acad. Sci. U.S.A. 97, 5022e5024. Persons, D.A., Hargrove, P.W., Allay, E.R., Hanawa, H., Nienhuis, A.W., 2003a. The degree of phenotypic correction of murine beta-thalassemia intermedia following lentiviral-mediated transfer of a human gamma-globin gene is influenced by chromosomal position effects and vector copy number. Blood 101, 2175e2183. Persons, D.A., Allay, E.R., Sawai, N., Hargrove, P.W., Brent, T.P., Hanawa, H., et al., 2003b. Successful treatment of murine b-thalassemia using in vivo selection of genetically-modified, drug-resistant hematopoietic stem cells. Blood 102, 506e513. Petersen, B.E., Bowen, W.C., Patrene, K.D., Mars, W.M., Sullivan, A.K., Murase, N., et al., 1999. Bone marrow as a potential source of hepatic oval cells. Science 284, 1168e1170. Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., et al., 1999. Multilineage potential of adult human mesenchymal stem cells. Science 284, 143e147. Porteus, M.H., Baltimore, D., 2003. Chimeric nucleases stimulate gene targeting in human cells. Science 300, 763.

Chapter | 77

Stem Cell Gene Therapy

Porteus, M.H., Cathomen, T., Weitzman, M.D., Baltimore, D., 2003. Efficient gene targeting mediated by adeno-associated virus and DNA double-strand breaks. Mol. Cell Biol. 23, 3558e3565. Prockop, D.J., 1998. Marrow stromal cells as stem cells for continual renewal of nonhematopoietic tissues and as potential vectors for gene therapy. J. Cell Biochem. Suppl. 30e31, 284e285. Qi, H., Aguiar, D.J., Williams, S.M., La Pean, A., Pan, W., Verfaillie, C.M., 2003. Identification of genes responsible for osteoblast differentiation from human mesodermal progenitor cells. Proc. Natl. Acad. Sci. U.S.A. 100, 3305e3310. Ramiya, V.K., Maraist, M., Arfors, K.E., Schatz, D.A., Peck, A.B., Cornelius, J.G., 2000. Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells. Nat. Med. 6, 278e282. Reyes, M., Dudek, A., Jahagirdar, B., Koodie, L., Marker, P.H., Verfaillie, C.M., 2002. Origin of endothelial progenitors in human postnatal bone marrow. J. Clin. Invest. 109, 337e346. Rivella, S., May, C., Chadburn, A., Riviere, I., Sadelain, M., 2003. A novel murine model of Cooley anemia and its rescue by lentiviralmediated human beta-globin gene transfer. Blood 101, 2932e2939. Russell, D.W., Hirata, R.K., 1998. Human gene targeting by viral vectors. Nat. Genet. 18, 325e330. Russell, D.W., Hirata, R.K., Inoue, N., 2002. Validation of AAV-mediated gene targeting. Nat. Biotechnol. 20, 658. Sadelain, M., Wang, C.H., Antoniou, M., Grosveld, F., Mulligan, R.C., 1995. Generation of a high-titer retroviral vector capable of expressing high levels of the human beta-globin gene. Proc. Natl. Acad. Sci. U.S.A. 92, 6728e6732. Sclimenti, C.R., Thyagarajan, B., Calos, M.P., 2001. Directed evolution of a recombinase for improved genomic integration at a native human sequence. Nucleic Acids Res. 29, 5044e5051. Sellers, S.E., Tisdale, J.F., Agricola, B.A., Metzger, M.E., Donahue, R.E., Dunbar, C.E., et al., 2001. The effect of multidrug-resistance 1 gene versus neo transduction on ex vivo and in vivo expansion of rhesus macaque hematopoietic repopulating cells. Blood 97, 1888e1891. Srivastava, A., 2002. Obstacles to human hematopoietic stem cell transduction by recombinant adeno-associated virus 2 vectors. J. Cell Biochem. Suppl. 38, 39e45. Stephan, V., Wahn, V., Le Deist, F., Dirksen, U., Broker, B., MullerFleckenstein, I., et al., 1996. Atypical X-linked severe combined immunodeficiency due to possible spontaneous reversion of the genetic defect in T cells. N. Engl. J. Med. 335, 1563e1567. Sutton, R.E., Wu, H.T., Rigg, R., Bohnlein, E., Brown, P.O., 1998. Human immunodeficiency virus type 1 vectors efficiently transduce human hematopoietic stem cells. J. Virol. 72, 5781e5788. Theise, N.D., Badve, S., Saxena, R., Henegariu, O., Sell, S., Crawford, J.M., et al., 2000. Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation. Hepatology 31, 235e240. Toneguzzo, F., Keating, A., 1986. Stable expression of selectable genes introduced into human hematopoietic stem cells by electric field-

949

mediated DNA transfer. Proc. Natl. Acad. Sci. U.S.A. 83, 3496e3499. Tsulaia, T.V., Prokopishyn, N.L., Yao, A., Carsrud, N.D., Carou, M.C., Brown, D.B., et al., 2003. Glass needle-mediated microinjection of macromolecules and transgenes into primary human mesenchymal stem cells. J. Biomed. Sci. 10, 328e336. Uchida, N., Sutton, R.E., Friera, A.M., He, D., Reitsma, M.J., Chang, W.C., et al., 1998. HIV, but not murine leukemia virus, vectors mediate high efficiency gene transfer into freshly isolated G0/G1 human hematopoietic stem cells. Proc. Natl. Acad. Sci. U.S.A. 95, 11939e11944. Van Damme, A., Vanden Driessche, T., Collen, D., Chuah, M.K., 2002. Bone marrow stromal cells as targets for gene therapy. Curr. Gene. Ther. 2, 195e209. Vassilopoulos, G., Navas, P.A., Skarpidi, E., Peterson, K.R., Lowrey, C.H., Papayannopoulou, T., et al., 1999. Correct function of the locus control region may require passage through a nonerythroid cellular environment. Blood 93, 703e712. Wada, T., Schurman, S.H., Otsu, M., Garabedian, E.K., Ochs, H.D., Nelson, D.L., et al., 2001. Somatic mosaicism in Wiskott-Aldrich syndrome suggests in vivo reversion by a DNA slippage mechanism. Proc. Natl. Acad. Sci. U.S.A. 98, 8697e8702. Wang, G., Seidman, M.M., Glazer, P.M., 1996. Mutagenesis in mammalian cells induced by triple helix formation and transcriptioncoupled repair. Science 271, 802e805. Wengler, G.S., Allen, R.C., Parolini, O., Smith, H., Conley, M.E., 1993. Nonrandom X chromosome inactivation in natural killer cells from obligate carriers of X-linked severe combined immunodeficiency. J. Immunol. 150, 700e704. Wengler, G., Gorlin, J.B., Williamson, J.M., Rosen, F.S., Bing, D.H., 1995. Nonrandom inactivation of the X chromosome in early lineage hematopoietic cells in carriers of Wiskott-Aldrich syndrome. Blood 85, 2471e2477. Woods, N.B., Muessig, A., Schmidt, M., Flygare, J., Olsson, K., Salmon, P., et al., 2003. Lentiviral vector transduction of NOD/ SCID repopulating cells results in multiple vector integrations per transduced cell: Risk of insertional mutagenesis. Blood 101, 1284e1289. Xiang, Y., Cole-Strauss, A., Yoon, K., Gryn, J., Kmiec, E.B., 1997. Targeted gene conversion in a mammalian CD34þ-enriched cell population using a chimeric RNA/DNA oligonucleotide. J. Mol. Med. 75, 829e835. Zielske, S.P., Gerson, S.L., 2002. Lentiviral transduction of P140K MGMT into human CD34(þ) hematopoietic progenitors at low multiplicity of infection confers significant resistance to BG/BCNU and allows selection in vitro. Mol. Ther. 5, 381e387. Zucali, J.R., Ciccarone, T., Kelley, V., Park, J., Johnson, C.M., Mergia, A., 2002. Transduction of umbilical cord blood CD34þ NOD/SCIDrepopulating cells by simian foamy virus type 1 (SFV-1) vector. Virology 302, 229e235.