E v o l u t i o n o f G e n e T h e r a p y,
`H i s t o r i c a l P e r s p e c t i v e
`
`Harry L. Malech, MDa,*, Elizabeth K. Garabedian, RN, MSLSb, Matthew M. Hsieh, MDc
`
`KEYWORDS
` Gamma retrovirus vector  Lentiviral vector  Gene editing
` Hematopoietic stem cells  Transduction  Apheresis  CD341 HSC
` Insertional mutagenesis
`
`KEY POINTS
` Gene therapy by the genetic modification of hematopoietic stem cells (HSC) has reached
`a stage of development that has resulted in substantial clinical benefits.
` This article explores the separate threads of knowledge, conceptual design, materials,
`and equipment required to reach our current era of clinically beneficial gene therapy.
` The history of gene therapy targeting hematopoietic stem cells include improvements in
`integrating vectors such as lentivectors and improvements in gene editing methods
`such as CRISPR/Cas9.
` Understanding the pathophysiology of adverse events such as insertional mutagenesis is
`important for seeking improvements in vector design that may enhance the safety of gene
`therapy.
`
`INTRODUCTION TO THEORETIC CONCEPTS AND EARLY BACKGROUND HISTORY
`IMPACTING HEMATOPOIETIC STEM CELL GENE THERAPY
`
`The history of gene therapy comprises the advance of theoretic concepts, under-
`standing of the human genome, availability of critical materials and instruments,
`design of vectors and chemical tools to manipulate and change genomic DNA, im-
`provements in the procurement and culture/maintenance of stemness of HSC in cul-
`ture, improvements in myeloid conditioning, the outcomes of conduct of clinical trials,
`observing successes and problems occurring in clinical trials, and deep study and
`
`Conflict of Interest: The authors declare no conflicts.
`a Genetic Immunotherapy Section, Laboratory of Clinical Immunology and Microbiology, Na-
`tional Institute of Allergy and Infectious Diseases, National Institutes of Health, 10 Center
`Drive, MSC1456, Bldg 10, Rm 5-3750, Bethesda, MD 20892-1456, USA; b National Human
`Genome Research Institute, NIH, 10 Center Dr, MSC1611, Bldg 10, Rm 10C-103, Bethesda, MD
`20892-1611, USA; c National Heart, Lung, and Blood Institute, NIH, 10 Center Drive, MSC1812,
`Bldg 10, Rm 9N119, Bethesda, MD 20892-1812, USA
`* Corresponding author.
`E-mail address: hmalech@niaid.nih.gov
`
`Hematol Oncol Clin N Am 36 (2022) 627–645
`https://doi.org/10.1016/j.hoc.2022.05.001
`0889-8588/22/Published by Elsevier Inc.
`
`hemonc.theclinics.com
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`elucidation of the mechanisms of problems that arise in clinical trials to seek and incor-
`porate corrective measures. The evolution of our understanding of ethical
`issues
`impacting gene therapy, and the logistics of access to and cost of successful gene
`therapy treatments are also important elements of this history.1
`In this broad brush and somewhat unconventional view of the history of gene
`therapy, we address general principles; key experiments, basic science, and clinical
`trials that illustrate some general principle; and the evolution of materials and instru-
`mentation that make current clinical approaches to gene therapy of HSC possible.
`We aim to complement rather than duplicate the extensive discussion of the back-
`ground studies of gene therapy and the march of the many published clinical trials
`in specific disorders or categories of disorders that are the subject of the other chap-
`ters in this series, as well as excellent recent reviews.2
`The earliest experiments that laid the foundation for gene therapy began with exper-
`iments on the transforming properties of bacteria.3 Alloway reported in 1932 that non-
`virulent (R type) pneumococci became lethal by adding cell-free extracts from virulent
`(S type) pneumococci. When injected with these “transformed” pneumococci, the
`mice developed pneumonia and died.
`In our view, the key conceptual background to all gene therapy emerged in the
`1940s with the seminal work by Avery and colleagues on bacterial transformation
`(which one could perhaps very loosely call gene therapy of bacteria). They identified
`DNA as the transforming factor that could change the physiology of a bacterial strain,4
`and more specifically, showed that the “transforming substance” was precipitated out
`by alcohol and later confirmed to be DNA. This was one of the key background ele-
`ments to Watson and Crick in identifying the structure of DNA,5 postulating its role
`as the genomic code of all prokaryotic and eukaryotic organisms, and thus demon-
`strating that nucleic acid sequences, rather than proteins, carry genetic information.
`The next critical discovery was that of Marshall Nirenberg, who in 1961 discovered
`the “triplet” code by which DNA encodes for the assembly of the 20 amino acids
`that serve as the building blocks of proteins.6
`In parallel with this elucidation of the biochemical basis of heredity, were emerging
`concepts from early transformation studies in mammalian cells, for example, the early
`reports that the transformation of 8-azaguanine sensitive cells with nuclei and chromo-
`somes from 8-azaguanine resistant cells rendered the transformants resistant due to
`transfer of a mutated hypoxanthine-guanine phosphoribosyltransferase gene.7,8 An
`early review of mammalian cell transformation studies conducted over the following
`18 years was reviewed in 1980 by Shows and Sakaguchi.9 This body of work further
`established that newly acquired biochemical traits from DNA transformation experi-
`ments in mammalian cells can be heritable.
`Many other key concepts that evolved into current methods of viral vector-
`mediated gene therapy were developed in the 1970s, during a period of the active
`investigation of viruses capable of transforming normal tissues into cancers. From
`this work, the concept emerged that perhaps these DNA and RNA tumor viruses
`known to insert into the genome of target cells could be modified in some way to
`remove the tumor causing elements, but retain their genome insertion capabilities
`to deliver a therapeutic payload. Some of the earliest published reviews of the history
`of gene therapy incorporating these essential concepts were those written in a series
`of reports over time by Theodore Friedmann10–13 who shared the 2015 Japan Prize
`with Alain Fischer for “For the Proposal of the Concept of Gene Therapy and its Clin-
`ical Applications.”
`More generally, the term “gene therapy” now broadly includes the introduction or
`manipulation of DNA or RNA sequences in human cells to treat disease. There is a
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`general consensus among the US Food and Drug Administration (FDA),14 the Euro-
`pean Medicines Agency (EMA),15 and the American Society of Gene and Cell Therapy
`(ASGCT)16 defining gene therapy as changes in gene expression, achieved by replac-
`ing or correcting a disease-causing gene, inactivating a target gene, or inserting a new
`or modified gene, using a vector or delivery system of genetic sequence or gene,
`genetically modified microorganisms, viruses, or cells.
`By the late 1970s, while our understanding of the molecular basis of human dis-
`eases was advancing through cloning and sequencing of genes, there were major
`technical challenges to implement gene transfer. Exogenous DNA could be introduced
`to target cells by transformation or transfection, but the overall efficiency was low.
`Additionally, if the introduced gene(s) did not provide a survival advantage, the dura-
`bility of gene transfer was also low. The resulting gene transfer efficiency at that time
`was about one in 100,000 cells, but nonetheless was proposed as a method to
`achieve genetic correction.17
`Intense interest in inherited hemoglobinopathies such as sickle cell disease and
`beta-thalassemia fueled work on beta-globin, one of the first genes to be cloned
`and then studied with the intent of gene transfer for clinical application. Mulligan
`and colleagues replaced the viral capsid protein (VP1) of the SV40 genome with
`complementary DNA of rabbit beta-globin in a monkey kidney cell line, which pro-
`duced large quantities of rabbit beta-globin mRNA and protein.18 As there was no
`inherent advantage for beta-globin gene transformed cells, several
`laboratories
`worked on selectable genes to be cotransferred. Pellicer and colleagues success-
`fully inserted beta-globin and thymidine kinase (TK) genes into murine teratocarci-
`noma cells.19 The Cline laboratory inserted dihydrofolate reductase (DHFR) or TK
`in murine marrow cells.20
`Cline and colleagues from UCLA then applied these results and tested them clin-
`ically.21–23 An experimental protocol to insert genetically modified marrow cells from
`patients with beta-thalassemia, inject the cells in the femur after local irradiation, and
`treat with a selecting agent was submitted to the human research review committee
`at their home institution. Because the first 2 patients to be treated were receiving
`their care in other countries (in a hospital in Naples, Italy and at Hadassah Hospital,
`Jerusalem, Israel), not covered by the UCLA review committee, the team sought in
`parallel and secured permission in Naples and Hadassah for the clinical study.
`Both patients were informed of the experimental nature and the low likelihood of suc-
`cess in this approach. After femur irradiation and infusion of modified marrow cells,
`the patients reported no adverse events, and selective agents were not used. Three
`months later, there was no demonstrable clinical benefit in both patients. Although
`safety of this clinical gene transfer was undebated, many controversial
`issues
`were brought forth.24–26 Can a clinical protocol proceed with permission from
`some but not all institutions? How many preclinical experiments (in vitro or animal),
`and what degree of “success” are needed to garner approval? While the responses
`to these issues are much more straightforward today, various review committees at
`that time were caught off guard and the consensus was that this was a rather prema-
`ture and in retrospect problematic initial attempt at the clinical application of gene
`therapy.27
`These first 2 attempts at human gene therapy generated much media attention and
`scrutiny by regulatory committees. The remainder of the decade into the early 1990s,
`scientists was quietly working on recombinant DNA methods, in vitro and animal
`models for testing, and strategies to enhance transgene expression. It quickly became
`clear that using viral vectors was more efficient in gene transfer than the previous
`methods of physical entry by transfection, fusion, or even electroporation. Much of
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`the gene transfer experiments then focused on vector optimization and design, and
`brought this background discussion into the early modern era of gene therapy.
`The following sections of this review will provide a historical background of a num-
`ber of parallel developments that provided the laboratory and clinical tools and mate-
`rials that facilitate our current approaches to gene therapy targeting blood cells
`including HSC.
`
`DESIGN OF INTEGRATING VECTORS USED FOR HEMATOPOIETIC STEM CELLS GENE
`THERAPY
`
`Vectors engineered from gamma retroviruses,28 long under study as the cause of a va-
`riety of cancers in mice, had the desired property of efficient insertion into the genome
`of target cells. Murine gamma retroviruses and their derivatives were the first of the
`genome integrating vectors to be applied to T lymphocytes and HSC in the clinical
`setting.
`Gamma retroviruses are RNA viruses, that on entry into a cell, are “reverse tran-
`scribed” (hence “retro”virus) into a DNA sequence. It is the DNA virus sequence
`that ultimately inserts itself into the host cell’s genomic DNA, becoming a “provirus”
`that in turn generates RNA virus sequences and viral mRNAs encoding virus proteins
`required for the replication phase of the virus life cycle. The critical issue was how to
`turn these viruses that efficiently insert provirus DNA genomic sequence into mamma-
`lian cell genomes, but are also efficient at causing tumors, into safe tools for gene ther-
`apy. The solution was to remove and/or inactivate as many elements of the virus
`genome as possible, while still retaining the ability of the highly engineered provirus
`sequence to insert efficiently into the mammalian cell genome. The goal was a func-
`tioning single-cycle virus capable of cell entry, uncoating, reverse transcription into
`provirus DNA, and insertion into the genome, but incapable of generating infectious
`virus. The solution involved separating the key elements required to generate
`replication-incompetent viral vector into 3 separate “production plasmids”: (1) an en-
`velope (env) producing element (the vector virus coat also serving the purpose of bind-
`ing to target cell and facilitating virus payload entry); (2) a gag-pol producing element
`(gag protein important for vector RNA packaging and polymerase for reverse tran-
`scribing the RNA); and (3) the vector sequence (retaining the psi element needed for
`packaging and the long terminal repeat (LTR) sequences at both ends of the vector
`sequence, which serves both as the internal strong promoter driving the production
`of a therapeutic protein and containing initiation elements binding the 2 ends of the
`vector for the circle formation required for reverse transcription). Where possible the
`env and gag-pol codons were changed to avoid recombination events that could
`reconstruct a replication-competent virus. To simplify the process of making different
`gamma retrovirus vectors, permanent packaging lines were devised that constitutively
`produce env and gag-pol, and when a specific vector sequence is added, clones
`could be assessed and chosen that constitutively produced vector in adequate titers.
`Many laboratories contributed to this technology and created a large array of different
`“flavors” of therapeutic gene therapy gamma retrovirus vectors. Many of these
`continue to be used for the production of some CAR-T lymphocytes or therapeutic
`cloned T cell receptors. This tour-de-force of engineering involving the contribution
`of many laboratories has served as the core technology used in the first generation
`of gene therapy targeting HSC or lymphocytes.
`The LTRs of gamma retroviruses were retained in the engineered vectors as conve-
`nient, very strong promoters to drive high levels of production of downstream inserted
`therapeutic protein-coding sequences. However, these same LTR elements contain
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`strong enhancer elements that can activate nearby genes. The engineered vectors by
`design retained the insertion targeting elements of the parent virus required to insert
`the DNA provirus into the mammalian genome. While the insertion of vector seems
`to be random, it is actually stochastic in that the mechanism used by the vector cou-
`ples to cellular elements, resulting in preferred sites of insertion into the genome.
`These preferred sites (also known as integration sites) are often located near the start
`of genes and in enhancer elements, and may in turn strongly interact with enhancer
`elements in the LTR.29,30 While the odds of any one insert occurring in a sensitive
`site are very low, gene therapy for a human subject may involve tens to hundreds of
`millions of insertions. Depending on the vector, the LTR and the host human subject
`disease substrate, we now know from adverse leukemic insertional mutagenesis
`events occurring in a number of clinical trials, that gamma retroviral vectors can trans-
`activate oncogenes such as LMO2, the MECOM complex, and other oncogene tar-
`gets to initiate the development of leukemia. These insertional mutagenesis events
`will be further discussed in greater detail in the last section of this historical review.
`Curiously, insertional mutagenesis leading to leukemic events has not been observed
`when the target of gamma retroviral vector gene therapy is T lymphocytes.
`Well before the first insertional mutagenesis, oncogenic events were observed in
`clinical trials of gene therapy using gamma retroviral vectors, certain limitations of
`this class of vectors (eg, limits of therapeutic payload size, limits on the use of alternate
`promoter elements instead of the LTR, absolute requirement for cell division for vector
`insertion into the genome) encouraged the development of gene therapy vectors
`derived from human immunodeficiency virus (HIV). HIV is part of a different group of
`retroviruses called lentiviruses and the vectors engineered from HIV are referred to
`here as “lentivectors.” HIV and other lentiviruses have a more complex structure,
`and have a number of required functional elements not present in gamma retroviruses,
`such as rev, that needed to be considered while engineering HIV into a safe gene ther-
`apy tool.31,32 As with gamma retroviruses, determining how much could be removed
`from the virus and whether the addition of elements from other viruses might enhance
`function and efficiency of the vector was an iterative discovery process. From a histor-
`ical perspective, some key advantages of lentivector function and engineering, and
`the insertional mutagenesis oncogenic events noted above have resulted for the
`most part in the abandonment of gamma retroviral vectors for the transduction of
`HSC for clinical trials.
`As with gamma retrovirus vectors, the production of lentivectors that are functional,
`but replication incompetent, required the separation of packaging elements into plas-
`mids separate from the transfer vector. Almost all lentivector production for clinical
`application uses the membrane fusion G protein derived from vesicular stomatitis virus
`(VSV-G) as the vector envelope element, rather than the natural env component of HIV.
`The cell membrane target of the VSV-G protein is ubiquitous to all cells with high ef-
`ficiency of binding and vector membrane fusion. Almost from the start, lentivector en-
`gineering strategies incorporated a self-inactivating (SIN) feature, modifying the LTR
`element that contains strong enhancers with transactivating potential and using safer
`promotors with little enhancer activity instead. This was accomplished by creating a
`0
`deletion in the 3
`LTR of the vector production plasmid. During vector production,
`0
`LTR assists in the important packaging biochemistry needed to produce
`the intact 5
`0
`infective but replication-incompetent lentivirus vector. During transduction, the SIN 3
`0
`LTR binds to the 5
`LTR in the circularization and priming step that retrotranscribes the
`0
`insertional provirus DNA from the lentivector RNA, and is incorporated into the 5
`end
`0
`LTR. This safety feature removes
`of the provirus DNA, thus “self-inactivating” the 5
`enhancer and activator elements, and allows the therapeutic payload transgene(s)
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`to be transcribed from a promoter of choice. The use of the SIN feature with an alter-
`nate internal promoter is not limited to lentivectors. A group of investigators incorpo-
`rated the SIN feature into the gamma retroviral vector that was efficacious in a clinical
`trial targeting hematopoietic stem cells (HSC) to treat infants with X-linked severe
`combined immunodeficiency (X-SCID). This SIN gamma retroviral vector showed clin-
`ical efficacy,33 with no insertional oncogenesis after median of 9 years of follow-up
`(personal communication S.-Y. Pai, 2022). Importantly, this maneuver does not alter
`the pattern of insertion characteristic of gamma retroviruses that tends to target
`0
`end of genes.33
`enhancer elements and the 5
`The larger payload capacity of lentivectors, SIN design, and potential to incorporate
`tissue-specific promoters and enhancers catalyzed concerted efforts by several in-
`vestigators to develop lentivectors that would drive high-level beta-globin expression
`at specific stages of erythroid precursor development. In the 1990s, the locus control
`region of beta-globin was discovered to contain hypersensitive sites (HS) that were
`important for high-level expression. After a series of in vitro experiments, a lentivector
`containing optimized regulatory elements, the TNS9 vector, was shown to drive high-
`level erythroid-specific expression of adult beta-globin in transduced murine HSC,
`successfully correcting a murine model of beta-thalassemia.34 This seminal work
`was followed shortly by the same group to correct another more severe phenotype
`of beta-thalassemia in Berkeley mice.35
`In parallel, another group designed a modified adult beta-globin for which the 87th
`amino acid was switched from threonine to glutamine, to mimic the antisickling effect
`of gamma-globin. This T87Q version successfully corrected 2 sickle mouse models36
`and was expressed at high levels when transduced human sickle cord blood stem
`cells were transplanted into immunodeficient mice.37 These reassuring preclinical
`studies ultimately led to the treatment of a patient with compound beta-E/beta-0 thal-
`assemia, who achieved transfusion independence.38
`From those early efforts, an increasing number of successful clinical trials have
`emerged to treat thalassemia and sickle cell disease, described in significant detail
`in other chapters in this series. Some of these studies will be revisited later in this
`article in comments relating to transduction enhancers and certain types of adverse
`events associated with gene therapy.
`
`GENE EDITING OF HEMATOPOIETIC STEM CELLS
`
`Gene editing is the most recently evolving technology to be applied to gene therapy for
`HSCs, having the capability of targeting a specific sequence within the genome, in
`contrast to the stochastic semirandom insertion of integrating viruses throughout
`the genome. The 4 main types of gene editing systems are meganucleases, zinc finger
`nucleases, transcription activator-like effector nucleases (TALENs),39 and clustered
`regularly interspaced short palindromic repeats-Cas (CRISPR-Cas).40 Of particular
`near term historical note are recent clinical reports of unequivocal clinical benefit
`from successful application of the CRISPR technology applied to HSCs for the correc-
`tion of hemoglobinopathies.41,42
`CRISPR is derived from an antivirus system that evolved in bacteria to “copy and
`memorize” virus sequence, thereby allowing the bacterium to target that same virus
`sequence for cleavage when subsequently infected by a similar virus.43 The discovery
`of the use of the CRISPR system for gene editing was the basis for the 2020
`Nobel Prize in Chemistry (https://www.nobelprize.org/prizes/chemistry/2020/press-
`release/). The CRISPR system has revolutionized the field, due to its ease of applica-
`tion and versatility. CRISPR-based derivative methods such as base editing and prime
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`editing use the core element of the CRISPR system to find the genome target
`sequence to enzymatically convert a single base pair or reverse transcribe a short
`sequence change into the genome, respectively.44
`Most current approaches to editing use electroporation to introduce the editing el-
`ements (CRISPR-Cas9 mRNA or protein, guide RNA, and any additional factors to
`augment editing) into HSC. There have been a number of small scale non-GMP
`research-grade electroporation instruments available for gene editing development
`work. Fortuitously, clinical scale and high throughput GMP compliant commercial in-
`struments in development in the past few years have become available just in time for
`the current initiatives in gene editing of HSC.
`
`HEMATOPOIETIC STEM CELLS: SOURCING, SELECTING, CULTURING, AND
`TRANSDUCING
`
`Parallel to the development of vectors and editing tools for gene modifying HSC, ad-
`vances in the procurement, culture, transduction, and engraftment of HSC have had
`an important impact on the field.
`HSC occupy niches in the marrow that facilitate the retention of pluripotent potential to
`give rise to all hematopoietic lineages and asymmetric proliferation of some progeny into
`lineage-specific progenitors.45 Sourcing HSC for gene therapy or conventional allogeneic
`transplants initially was restricted to the harvesting of bone marrow with needles. That HSC
`are constantly translocating at a slow rate from marrow to the circulation and back to the
`marrow was a key discovery that ultimately led to alternate sourcing of HSC.46 There is a
`steady state presence of CD341 HSC or progenitors in the peripheral blood of healthy
`humans of about 1400 cells per ml (1.4 per ml). This low baseline frequency of CD341
`HSC and progenitors in the circulation can be increased by daily injections of granulocyte
`colony-stimulating factor (G-CSF or filgrastim), which induces a transient release of
`CD341 HSC and progenitors from the marrow into the peripheral blood, peaking at 5 to
`6 days at an average of 76 per ml (a 50-fold increase), then declining, even with additional
`daily injections.47,48 Subsequent studies indicated that CXCR4 (the receptor for stromal
`cell-derived factor-1 [SDF-1], also known as CXCL12) tethers HSC within the marrow
`and that G-CSF breaks that tether by increasing granulocyte proliferation and release of
`granule enzymes in the marrow, thereby enhancing the release of HSC into the circulation.
`More recently a small molecule inhibitor of the binding site of CXCR4, plerixafor (previously
`called AMD3100), was also shown to release HSC from the marrow to the circulation,49
`and when administered in the combination with G-CSF results in a synergistic mobilization
`of HSC to the peripheral blood.50
`The ease of apheresis collection of stem cells using continuous flow instruments
`following mobilization has resulted in this method becoming the preferred sourcing
`of HSC for many gene therapy clinical studies. Some patients with some inherited
`blood disorders that impair marrow proliferation (Fanconi anemia for example) will
`not mobilize, and infants who are too small for standard apheresis procedures still
`require bone marrow aspiration for sourcing HSC for gene therapy. Patients with sickle
`cell disease are at high risk of adverse events including vaso-occlusive crises when
`treated with G-CSF. Fortunately, several groups have shown that efficient and safe
`mobilization of HSC from marrow to peripheral blood can be accomplished using
`plerixafor alone as the mobilization agent in patients with sickle cell.51–54
`
`Apheresis
`
`The first apheresis device for separating blood components was developed in a
`collaboration between the National Cancer Institute and IBM,55 and was appreciated
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`at the time as a critical breakthrough in instrumentation for the separation and collec-
`tion of blood components.56 A number of companies developed a series of progres-
`sively efficient continuous flow instruments to harvest of fractions enriched in HSC
`from G-CSF and/or plerixafor mobilized donors. Without this instrumentation now
`commonplace in blood collection centers, and simply viewed by the gene therapy
`community as “background” standard banking technology, much of the current prog-
`ress in gene therapy would not have been possible.
`
`Selection of Hematopoietic Stem Cells from Marrow or Apheresis Products
`
`Multipotent permanently repopulating HSC express the CD34 surface antigen that
`was originally called My-10 as detected by a murine monoclonal antibody, raised
`against the KG-1a human tumor cell line.57 Studies later demonstrated that immuno-
`magnetic beads coated with anti-CD34 antibody could form complexes with HSC in a
`marrow or apheresis product, which in turn could be purified with magnets. These
`initial studies used incubation with chymopapain to release HSC from the beads.58
`This magnetic bead selection approach became the basis for the commercial devel-
`opment of instruments for selective enrichment of HSC, 2 of which reached the late
`commercialization phase.
`Baxter International, Inc (then called Baxter Healthcare Corporation) together with
`its “spin-off” subsidiary Nexell, Inc was the first to develop a fully automated instru-
`ment called the Isolex 300i that relied on the binding of HSC to magnetic beads coated
`with the anti-CD34 antibody. Following magnetic separation, the washed product was
`exposed to an octapeptide that directly competed for the binding site of the anti-CD34
`antibody to the CD34 antigen, thus removing the antibody complexed magnetic beads
`from the cells. This allowed the beads to be retained by a second pass through the
`magnetic field, yielding a cell product free of the beads and antibody.59 A precommer-
`cial manual version of the Isolex system was used in one of the earliest clinical studies
`of gene therapy for CGD.60
`A very similar system developed in parallel by Miltenyi Biotec GmbH became their
`CliniMACS CD34 Reagent System. This system uses anti-CD34 antibody chemically
`conjugated to dextran beads with an iron oxide/hydroxide core. A binding column
`with a magnetic gradient is used to separate the HSC bound to the anti-CD34 mag-
`netic dextran bead from marrow or apheresis product. Unlike the Isolex system, there
`is no maneuver to separate the cells from the antibody-conjugated beads, which are
`presumably degraded in culture or in vivo following transplantation. In October 2003
`exclusive rights to market, the Isolex system was acquired by Miltenyi and not further
`developed by them leaving only the CliniMACS and its derivative devices available
`currently for the clinical selection of HSC for gene therapies.61
`
`Culture and Transduction of Hematopoietic Stem Cells
`
`As noted previously, successful gamma retrovirus vector transduction requires 1 cell
`division cycle to complete integration into the genome of a cell. While lentivector trans-
`duction does not absolutely require cell division, HSC must enter at least the G1 phase
`of the cell cycle, and exposure of HSC to growth factors for a period of time in culture
`seems to enhance transduction efficiency. While it was initially hoped that gene editing
`methods might not require the activation of HSC, a similar improvement of gene edit-
`ing when HSC are cultured with growth factors has been observed by many investiga-
`tors. Defining ex vivo culture conditions and growth factor combinations that enhance
`transduction or editing while minimizing loss of long-term marrow repopulating poten-
`tial has been intensely studied. The discovery of each of the many critical HSC growth
`factors important for both efficient vector transduction and gene editing approaches
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`will not be reviewed here; suffice it to say that without the work to identify, clone, and
`provide GMP compliant growth factors, gene therapy for HSC could not have
`advanced. While there is no consensus about “best” conditions, the minimum combi-
`nation of three growth factors, stem cell factor (SCF), FLT3-L, and thrombopoietin
`(TPO) is used by most of the investigators for clinical gene therapy for HSC applica-
`tions. Additional factors used by some investigators include interleukin (IL)-3 and/or
`IL-6. A key limiting issue is that culture in these growth conditions beyond 3 to
`4 days results in a significant loss of long-term repopulating potential. Outside the
`scope of this review is the discovery of other biochemical factors and conditions
`that may prolong the period during which HSC can be maintained in culture while
`delaying the loss of long-term engraftment potential. This is an important emerging
`field for the future impact on HSC gene therapy.
`While culture and transduction of HSC may be performed in standard tissue culture
`flasks, gas permeable flexible plastic culture bags are increasingly used to achieve
`more “closed system” handling. The earliest application of such systems in gene ther-
`apy clinical trials suggests better gas exchange, more consistent high viability and
`yield, and transduction efficiency.60,62
`
`Transduction Enhancers
`
`Maneuvers to achieve the highest efficiency transduction by gamma retroviral and len-
`tiviral vectors have been an important aspect of the history of gene therapy. Quite early
`on, it was shown that the addition of certain charged polymers to the transduction cul-
`ture, such as polybrene or protamine sulfate, would enhance transduction by gamma
`retrovirus vectors; of the 2, protamine sulfate continues to be used to enhance the
`transduction of lentivectors.63 Physical maneuvers such as centrifuging the culture
`plate or gas permeable bag have also been shown to enhance transduction,64 but
`the practicalities of application at clinical scale have limited the translation of this ma-
`neuver into the clinic. It has been presumed but not proven that ionic polymers and
`centrifugation worked in par