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Transfer of genes into HSCs could be used to treat a variety of diseases from AIDS to cancer. HereBordignon and Roncarolo discuss the logistics and progress of HSC gene transfer technology for

the treatment of different diseases.

Therapeutic applications forhematopoietic stem cell gene transfer

Claudio Bordignon and Maria Grazia RoncaroloHSR–Telethon Institute for Gene Therapy (TIGET) of Genetic Disease, Cancer Immunotherapy and Gene Therapy Program, Istituto Scientifico H.S. Raffaele, Universitá Vita-Salute San Raffaele, via Olgettina 58, 20132 Milan, Italy. (claudio.bordignon@hsr.it)

Over the last two decades, it has been proposed that the transfer ofgenes into hematopoietic stem cells (HSCs) could be a tool for thetreatment of genetic diseases as well as a number of acquired dis-eases, including cancer, AIDS and autoimmune and neurodegenerativedisorders. With the clinical application of gene therapy progressing inparallel with a better understanding of stem cell biology, the numberof different target cells, tissues and organs used in gene therapy hasincreased significantly. However, HSCs remain the main cellular tar-get for genetic intervention in a number of clinical settings of primarymedical and scientific relevance, especially, but not only, those thataim to correct or modulate the immune system.

This commentary will briefly examine the gene transfer technolo-gies used for ex vivo transduction of HSCs; this will be followed bya short description of the logistics required for ex vivo transductionand its impact on the diffusion and commercialization of gene-trans-fer products. How the available tools are used to transfer genes intoHSCs will be discussed with regard to the treatment of congenitaland acquired immunodeficiencies as well as other disorders, includ-ing autoimmunity, that are caused by an imbalance of the immunesystem. Cancer gene therapy will also be addressed from the per-spective of immuno-gene therapy, which includes gene-transferstrategies aimed at eradicating cancer cells by inducing tumor-spe-cific effector T cells.

Viral vectors for HSC gene transferAll viral and synthetic vectors developed for different gene therapymodels have been tested for their ability to transduce HSCs; most ofthe data have been generated with viral vectors, predominantly murineonco-retroviruses1. These vectors permanently integrate the trans-ferred gene into the genome of the host cell, which should maintaintransgene expression during proliferation, differentiation and matura-tion in all the cell lineages. However, target cell division is requiredfor gene transduction with these vectors, whereas immature HSCs arenaturally quiescent. To overcome this initial obstacle, a large body ofresearch focused on inducing cell division while preserving the self-renewal ability of stem cells and their potential to expand and differ-entiate into all blood lineages. Strategies involved the use of differenthematopoietic growth factors in various combinations, with or withoutthe additional use of bone marrow stromal cell layers and humanrecombinant fibronectin fragments. The abilities of these strategies tofavor gene transfer efficiency while preserving the “stem cell poten-tial” of the transduced population were explored. This massive effortresulted in significant progress in the development of animal modelsof HSC gene transfer. However, for several years this research had noimpact on gene therapy for human diseases. Data on the feasibility ofstem cell gene therapy in humans only became available a few years

ago in a small number of clinical settings, including severe-combinedimmunodeficiencies (SCIDs) and several gene-marking studies in thecontext of bone marrow transplantation. But either the clinical rele-vance of these results was difficult to evaluate or transgene expressionwas below the threshold required for a clinical benefit.

The only demonstration that stem cell gene therapy could clinicallyrepair a genetic defect came from studies in children with SCIDs (seebelow). However, functional cells of lymphoid lineage have such astrong selective advantage that even an extremely low number oftransduced cells have the potential, over time, to expand in vivo andreplenish the “empty space” left by the missing elements of theimmune system. Thus, proof that stem cell gene therapy can perma-nently transduce cells of all the hematopoietic lineages is withinreach, but remains to be shown. The combination of myeloablation,improved gene transfer protocols and the superior gene transfer effi-ciency of new viral vectors have shown this potential both in primatemodels2 and in clinical studies of SCIDs. Longer follow-ups of theclinical trials and broader applications—such as the use of gene trans-fer protocols in hemoglobinopathies or lysosomal storage disorders—are needed to confirm these achievements. In addition, it should beremembered that at the moment stem cell gene therapy is strictlydependent upon ex vivo manipulation, a complex procedure that willrestrict its implementation to highly specialized medical centers, sig-nificantly limiting its widespread use.

The discovery and development of new vectors derived from HIVsharply accelerated progress in the field of HSC gene transfer. Thisfamily of lentiviral vectors can transduce dividing and nondividingcells from different lineages, tissues and organs, including HSCs,with great efficiency3. Lentiviral vectors efficiently transduce nondi-viding cells after direct in vivo injection, thus making genetic engi-neering easier and more suitable for large-scale medical application.However, safety concerns related to the use of vectors derived from ahuman pathogen and technical problems associated with large-scaleproduction and validation have hindered their application as a newgene transfer tool. As yet, no clinical protocol for their use inpatients has been approved.

In some diseases—such as hemoglobinopathies or SCIDs—direct invivo gene transfer requires that expression of the transferred genes beconfined to the lineage of interest. Targeted transduction could theoret-ically be achieved by manipulating the vector envelope, so that bindingand infection is confined to a specific cell lineage. However, this formof targeting (or, conversely, detargeting) has proven ineffective so far4.Another approach—the insertion of regulatory sequences into the vec-tors to control transcription and confine expression of the transgene toa specific lineage after cell differentiation—looks more promising.This strategy, called transcriptional targeting4, and related approaches

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have already been used successfully in preclinical models to confineglobin expression to the erythroid lineage after HSC transduction5.

Congenital and acquired immunodeficienciesIn July 1990, gene therapy of the adenosine deaminase–deficient vari-ant of SCID (ADA-SCID) was, together with immuno-gene therapyof melanoma, the first clinical trial approved by the NationalInstitutes of Health Recombinant DNA Advisory Committee. Bothprotocols included gene transfer into lymphoid cells. Despite therationale supporting HSC gene therapy for the permanent correctionof the metabolic and immune defect associated with ADA-SCID, itwas concluded at the time that lymphocytes were an easier transduc-tion target. This trial was initiated in September 1990 and was fol-lowed 2 years later by the first HSC gene therapy study for the samedisease6. Other studies—including gene transfer into cord blood stemcells7—followed in the USA, Europe and Japan. Clear indications ofthe persistence of genetically engineered cells derived from trans-duced long-living lymphocytes and transduced HSCs were obtainedfrom these studies. However,the availability (and concomi-tant use in all patients) of analternative treatment—pegilat-ed bovine enzyme (PEG-ADA) replacement therapy—made it difficult to assess thetherapeutic effect of gene ther-apy, so much so that genetherapy was repeatedlyreferred to as a “failure” in thetreatment of ADA-SCID,despite the clear demonstra-tion that the use of a combina-tion of gene and enzyme ther-apy was superior, in somepatients, to the use of PEG-ADA therapy alone6. In twoseparate studies, the use ofPEG-ADA was discontinuedand led to partially differentconclusions. In the cord bloodstem cell gene therapy study,enzyme discontinuation result-ed in the accumulation of Tlymphocytes with a normalADA gene, but immune functions were not sustained8. However, in theother study—in which lymphocytes were transduced—the geneticallymodified T cells progressively replaced the nontransduced population.Proliferative responses to polyclonal stimulation were fully restoredand vaccination with T cell–dependent neo-antigens induced primaryand secondary antibody production with immunoglobulin G (IgG)class switching (A. Aiuti et al., unpublished data).

Despite promising data from animal or in vitro models of othercongenital immunodeficiencies—for example, Jak3-deficient SCID,WAS (Wiskott-Aldrich Syndrome) and RAG (recombination-activat-ing gene) deficiency9—the clinical application of stem cell gene thera-py has been limited to chronic granulomatous disease10 and SCID-X111. However, these studies have helped the field progress. Forexample, results from the SCID-X1 gene therapy trial provided thefirst formal demonstration that stem cell gene therapy alone couldcure a genetic disease11. These important results were achieved using

a combination of cytokines (including stem cell growth factor, throm-bopoietin, interleukin 3 (IL-3) and Flt3 ligand) in the ex vivo trans-duction protocol, and thanks to the strong selective advantage oftransduced progenitor cells that, in the absence of functional T cells,favored expansion along the T cell lineage11.

Based on these results, a similar protocol was recently used in thetreatment of ADA-SCID with stem cell gene therapy. In contrast togene therapy in the treatment of SCID-X1, phenotypic correction ofADA-SCID could only be achieved after mild myeloablation [Aiuti,A. et al. Blood 98, abstract 780, p. 3246 (2001)], which suggestedthat in vivo selective advantage of transduced HSCs may differdepending on the SCID variant. In ADA-SCID, the immune deficitrepresents the dominant component of a more complex phenotypecaused by a metabolic alteration that also affects other lineages andorgans, including the kidney and liver. Thus, it seems likely that oneof the two alternative strategies—transduction of stem cells orperipheral blood lymphocytes—should be chosen carefully based onthe phenotype of the particular genetic defect (Fig. 1).

The results of gene therapytrials for various SCIDs havesuggested that a similarapproach could be useful inthe treatment of AIDS. TheHIV life cycle has been exten-sively studied, which meansthat a number of candidategenes are available that couldbe used to block replicationand spreading of the virus.These genes include thoseencoding trans dominant-neg-ative mutants, decoysequences for regulatory pro-teins and antisense RNAs12.Therapy that uses these genesmust be evaluated in con-junction with conventionaltreatments and should takeinto account the clinical ben-efit presently provided bymultidrug combination thera-pies and the new biologicaland immunomodulatoryapproaches. However, recent

progress in our understanding of both the nature of HIV-1 infectionand the major toxicity associated with lifelong highly active anti-retroviral therapy (HAART), highlight the need to develop alterna-tive therapeutic strategies aimed at long-term control of HIV, with orwithout the use of HAART13.

Clearly, the treatment of AIDS by gene therapy will pose difficultchallenges; the efficient transduction of stem cells and the restrictedexpression of protective transgenes in T lymphocytes, dendritic cells(DCs) and macrophages must be achieved in the presence of a func-tional thymus that can support adequate T cell differentiation. An alter-native would be to combine stem cell and lymphocyte gene therapy.This may allow preservation of the existing immune repertoire whilethe genetically engineered HIV-resistant lymphocytes progressivelydifferentiate and expand.

The use of HIV-derived lentiviral vectors for gene therapy in HIV-infected patients has been proposed as the safest setting for initially

Figure 1.The advantages, limitations and risks associated with stem cellversus lymphocyte gene therapy in the treatment of ADA-deficient SCID.The main advantages conferred by the use of stem cell gene therapy in the treatmentof a genetic disease of the hematopoietic system are shown.Although several aspectsare unique to this disease model, the results can be extended to other SCID variantsand, to lesser extent, to other hematological diseases.

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testing these vectors in humans14. However, concerns have been raisedabout the possibility of recombination events between the vectorsequences and wild-type HIV occurring in patients, thus generatingnew variants of the virus with unpredictable biological properties.Although extremely unlikely, this possibility should be kept in mindwhen evaluating the benefits and potential risks associated with theuse of lentiviral vectors derived from HIV in AIDS gene therapy.

Immuno-gene therapy of cancerCancer, in particular tumors treated by autologous bone marrowtransplantation, was one of the first clinical applications for HSCgene transfer technology. Initially the main objectives of gene-mark-ing studies were to define the origin of tumor relapse (graft or recipi-ent) and the efficiency of stem cell gene transfer15. The relevance ofthese studies to the progress of the field is due to the similaritiesbetween the gene transfer protocols used for the transduction ofHSCs in this clinical setting and those required for successful genetherapy of congenital immunodeficiencies. Over the years followingthese first attempts, the use of gene therapy in the treatment of can-cer expanded to become the main preclinical and clinical applica-tions for gene therapy. Transfer of drug-resistance genes into HSCs,combined with dose intensification of chemotherapeutic agents, hasbeen applied to autologous bone marrow transplantation in order totreat various tumors. These studies were directly derived from theabove-mentioned gene-marking studies, but had limited success.However, the combined in vivo use of drug-resistance genes andtherapeutic genes may be relevant for clinical applications in whichno selective advantage has been anticipated.

The majority of immuno-gene therapy approaches for the treatmentof cancer are focused on the use of gene transfer to increase theimmunogenicity of tumor cells16. It is believed that, independent of theapproach used to debulk the tumor mass (for example, chemo- or radio-therapy, surgery or anti-angiogenic therapy), the immune system will

have an important function in eliminating the residual tumorcells, thus playing a crucial role in the final outcome of theantitumor therapy. Immuno-gene therapy includes the transferof genes encoding the major histocompatibility complex(MHC), B7 and other costimulatory molecules, cytokines(including IL-2, IL-4, interferon-γ, tumor necrosis factor-α(TNF-α), IL-7, IL-12 and granulocyte-macrophage–colony-stimulating factor) and chemokines into tumor cells to makethem into professional antigen-presenting cells (APCs)16.Using this approach, important progress has been made in ani-mal models, but its efficacy in humans has yet to be demon-strated. The apparent clinical failure of this technique is due toa number of reasons. First, too many clinical trials have beendesigned as phase I studies (which evaluate toxicity only). Insuch studies, patient accrual has been systematically limited toadvanced or end-stage patients who have been heavily pretreat-ed with chemotherapy and radiations. This dramaticallyreduces any chance to induce an efficacious tumor-specificimmune response. It is also possible that human tumors aremuch less immunogenic than the tumor models used in pre-clinical studies. However, better designed proof-of-conceptphase II studies, in which immunotherapy is used as a first lineof treatment, are mandatory in order to fully evaluate the clini-cal potential of cancer immuno-gene therapy.

Gene transfer into HSCs is now a major player in the fieldof cancer immuno-gene therapy through the production ofbone marrow–derived DCs (BMDCs) that are genetically

engineered to express tumor-associated antigen (TAA) ex vivo or invivo (Fig. 2). DCs, which play a central role in shaping the immuneresponse17, are currently being actively exploited in cancer immuno-gene therapy18. Genetic engineering of autologous BMDCs may haveseveral advantages, such as permanent and sustained antigen expres-sion compared with other forms of tumor antigen–loading and theestablishment of a persistent antitumor response. Because mature DCscannot divide, the two processes of tumor antigen gene transfer and invitro generation of BMDCs can be combined to produce professionalAPCs that are able to induce tumor-specific cytotoxic T cells.

Genetic manipulation of autoimmune disordersThere is great potential for the development of more efficacious andspecific gene therapies for autoimmune disorders. The knowledgeacquired from the study of autoimmune disease pathogenesis has ledto the development of several gene therapy approaches in recentyears, including the modulation of cytokine responses to reprogramthe immune system, delivery of anti-idiotypic vaccines and the rein-duction of tolerance. The rationale for modulation of cytokine signal-ing is based on the involvement of inflammatory cytokines, such asIL-1, IL-12 and TNF-α, as well as T helper type 1 (TH1) cells inautoimmune diseases19. In an attempt to shift the balance toward lesssevere autoimmune responses, viral vectors or naked DNA that deliv-er anti-TH1 and/or TH2 cytokines with or without inhibitors of specif-ic cytokine receptors have been tested in animal models of multiplesclerosis, lupus and diabetes20. Delivery of anti-inflammatory andsuppressive cytokines, such as TGF-β or IL-10, to the site of aggres-sion has also been successfully applied to several models of autoim-mune disease, including diabetes21. The major challenges faced bythese approaches, however, are the difficulty of inducing an antigen-specific effect, as opposed to a broader state of immunosuppression,and the concern that a cytokine imbalance may perturb other immuneresponses in the treated host.

Figure 2. The gene transfer protocol used to genetically engineer DCs toachieve permanent tumor antigen presentation. The simultaneous expansion ofhematopoietic stem cells to obtain DCs in vitro and the transduction procedure allowstransfer of the TAA gene by onco-retroviral vector machinery that requires the target cellto undergo active cell division.

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The use of anti-idiotypic vaccines could provide a more specificmethod for gene therapy intervention in autoimmunity, which wouldeliminate or control autoreactive cells. In T cell–mediated autoimmunediseases, particular T cell receptor (TCR) variable (V) gene segmentsare often overexpressed by the pathogenic T cells. For example, Vβ

expression by myelin basic protein–specific T cells from patients withmultiple sclerosis is skewed towards Vβ5.2 and Vβ6.122. Therefore, theinduction of an anti-idiotypic response via immunization with an anti-gen to the TCR may be one strategy with which pathogenic T cellclones can be suppressed. This type of immunization can be achievedeither by the injection of synthetic peptides or through gene therapy23,which is more efficient.

For many autoimmune diseases, gene therapy could potentially beused to insert a new gene or correct a defective gene. For example, inrheumatoid arthritis, the gene encoding Fas ligand can be inserted inthe synovial cells to induce localized apoptosis24, whereas in patientsaffected by lupus erithematosus, the known defect in the C1q com-plement fraction can be targeted25. Finally, gene therapy can be usedto reinduce tolerance in an autoimmune patient and to generate apool of regulatory T cells able to maintain immunological homeosta-sis. In addition to its potent anti-inflammatory activity, IL-10 is acrucial factor in the differentiation of T regulatory type 1 cells26.Therefore, transduction of antigen-specific T cells with the geneencoding this cytokine could lead to the differentiation of a pool ofregulatory T cells able to suppress the pathogenic responses.

In most of the experimental approaches mentioned so far, genetransfer vectors, both viral and nonviral, are widely used to transducemature immune cells, such as T cells and DCs, or target tissues.However, the use of HSCs or immature progenitors as a target forgene therapy may have several advantages and allow the developmentof new therapeutic strategies. Indeed, the expression of an antigen inbone marrow hematopoietic precursors should lead to the induction oftolerance specific for that antigen. This principle has been demon-strated for viral and MHC antigens27, but it could also be applied toautoantigens such as insulin, GAD (glutamic acid decarboxylase) andIA-2, which are the major autoantigens involved in diabetes.Transduction of CD34+ cells with vectors containing genes encodingautoantigens under the control of tissue-specific promoters can lead toexpression of these autoantigens in the thymus with the consequentinduction of central tolerance. Specifically, overexpression ofautoantigens in DC precursors that migrate to the thymus can result inthe negative selection of autoreactive T cell progenitors. Indeed, dur-ing thymic maturation, CD4+ and CD8+ lymphoid progenitors thatrecognize autologous antigens presented in the context of MHC classI or II by thymic APCs undergo apoptosis and are clonally deleted.This approach combined with peripheral T cell ablation could com-pletely “reboot” the immune response. On the other hand, transduc-tion of HSCs with genes encoding IL-10 and TGF-β under the controlof a promoter specific for the targeted tissue may lead to a specificimmunosuppression at the site of inflammation, in a similar mannerto that described above. The advantage of using stem cells, rather than

mature T cells or DCs, is that transduced stem cells self-renew in thebone marrow and therefore provide a permanent “correction”.

In correcting the damage caused by autoimmune aggression—such as β cell disruption in insulin-dependent diabetes—gene thera-py could potentially restore the β cell function by inserting theinsulin gene in alternative tissues where it can be expressed28.However, such a strategy will hardly provide the tight control ofglucose concentrations that is supplied by glucose-dependant rapiddegranulation as a result of insulin accumulation in β cell–specificgranules. Genetic manipulation may provide an alternative approachby controlling transdifferentiation of stem cells into insulin-produc-ing β cells. This has already been achieved with embryonic stemcells29. However, the capacity of HSCs to transdifferentiate into mul-tiple cell lineages means that the genetic engineering of these cellsto generate functional β cells can also be envisioned.

The future of stem cell gene therapyHSCs will remain a crucial target for gene therapy. The impact, dimen-sion and speed at which clinical application will proceed depend upon afew crucial variables. The ability of the new generation of vectors toshorten the culture time, while increasing the efficiency of transduction,relies heavily upon the progress in lentiviral vector technology.Improving the packaging efficiency is a prerequisite for safety and effi-ciency. The development of vectors based on lentiviruses other than HIVcould also significantly increase their safety. In addition, direct deliveryof lentiviral vectors in bone marrow may be efficacious in transducingHSCs in vivo. Finally, controlling the specific signals that lead to self-renewal of stem cells versus differentiation into mature hematopoieticcells or transdifferentiation into mature cells of other tissues, will play adecisive role in shaping this field of gene and cellular therapies.

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