toh 18 gene therapy revised 2008

8/3/2019 TOH 18 Gene Therapy Revised 2008

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T R E A T M E N T O F H E M O P H I L I AAPRIL 2008 NO 1

GENE THERAPY FOR THEHEMOPHILIAS

Third Edition

David LillicrapQueens UniversityOntario, Canada

Arthur R. ThompsonUniversity of Washington and the Puget Sound Blood CenterWashington, U.S.A.


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Published by the World Federation of Hemophilia (WFH), 1999; revised 2004, 2008.

World Federation of Hemophilia, 2008

The WFH encourages redistribution of its publications for educational purposes by not-for-profithemophilia organizations. In order to obtain permission to reprint, redistribute, or translate thispublication, please contact the Communications Department at the address below.

This publication is accessible from the World Federation of Hemophilias website at www.wfh.org.Additional copies are also available from the WFH at:

World Federation of Hemophilia1425 Ren Lvesque Boulevard West, Suite 1010Montral, Qubec H3G 1T7CANADA

Tel. : (514) 875-7944Fax : (514) 875-8916E-mail: [email protected]: www.wfh.org

The Treatment of Hemophilia series is intended to provide general information on the treatment andmanagement of hemophilia. The World Federation of Hemophilia does not engage in the practice ofmedicine and under no circumstances recommends particular treatment for specific individuals. Doseschedules and other treatment regimes are continually revised and new side effects recognized. WFHmakes no representation, express or implied, that drug doses or other treatment recommendations in thispublication are correct. For these reasons it is strongly recommended that individuals seek the advice of amedical adviser and/or consult printed instructions provided by the pharmaceutical company beforeadministering any of the drugs referred to in this monograph.

Statements and opinions expressed here do not necessarily represent the opinions, policies, orrecommendations of the World Federation of Hemophilia, its Executive Committee, or its staff.

Treatment of Hemophilia MonographsSeries EditorDr. Sam Schulman


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Table of Contents

Summary..................................................................................................................................................................... 1

Introduction................................................................................................................................................................ 1

Vectors for Gene Transfer......................................................................................................................................... 2Retroviral vectors ........................................................................................................................................ 2Table 1: Viral Vectors Used for Preclinical Gene Transfer of Factors VIII or IX ................................. 2Adenoviral vectors ...................................................................................................................................... 3Adeno-associated viral (AAV) vectors ..................................................................................................... 3Non-viral vectors......................................................................................................................................... 4Alternative cell types .................................................................................................................................. 4Gene repair................................................................................................................................................... 4

Human Trials of Hemophilia Gene Transfer ......................................................................................................... 4Hemophilia A............................................................................................................................................... 5Table 2: Clinical Trials of Gene Transfer for Hemophilias A or B ........................................................ 6Hemophilia B ............................................................................................................................................... 7

Human Safety Considerations with Viral Vectors ................................................................................................ 7Retroviral vectors .........................................................................................................................................7Adenoviral vectors ...................................................................................................................................... 8Adeno-associated viral vectors.................................................................................................................. 8Patient participation.................................................................................................................................... 8

Conclusion.................................................................................................................................................................. 9

Cited References ........................................................................................................................................................ 9

General References .................................................................................................................................................. 12


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Gene Therapy for the Hemophilias

David Lillicrap, Arthur R. Thompson

Summary

Clinical trials of gene transfer in the hemophiliasevolved from a decade of studies in culturedcells and then in animal models. The limitedsuccess from the initial human experience hasprovided focus on a need for improvements inor alternatives to the viral vector transfersystems used in subjects with hemophilia A orB. Several laboratories have modified vectors toimprove expression of these clotting factors,while considerable effort is aimed at improvingsafety. Although a variety of new approachesare promising, a clearly improved strategy hasyet to emerge.

Incorporation of intron and other elements intothe factor VIII or IX transgene improves theamount of factor expression in each cell. Tissue-specific promoters limit expression in othertissues including immune cells, to reduce therisk of developing inhibitors. Retroviral vectors,the ones originally introduced for gene transfer,are more successful in neonatal mice; the relatedlentiviral vectors will transduce non-dividingcells, although the long-term safety of either

remains a concern. Adenoviral vectors strippedof their viral genes have fewer complications,but there is still the need to develop a saferdelivery envelope. Different serotypes andmodified adeno-associated viral vectors increaseexpression and allow inclusion of the largerfactor VIII genes. Effective delivery systems fornon-viral vectors in vivo require developing;nevertheless, a rodent model demonstrates thatlong-term expression of therapeutic levels offactors VIII or IX can be realized without a viralvector. Although the primary target cell for gene

transfer remains the liver, there is progress withexpression from a variety of other tissues.Strategies for tolerance induction or preventionof immune responses to expressed factors VIIIand IX are emerging to improve the safetyprofile and developments in understanding theimmune response, especially to factor VIII, areaddressing this potentially severe complication.Finally, efforts of gene repair are showing initialprogress, especially with a trans-splicing

approach that corrects the hosts own mutant

messenger RNA, allowing synthesis of a normalprotein even in the absence of the normal genesequence.

Introduction

For a patient with hemophilia, gene therapywould allow continuous synthesis of a normalprotein to correct the deficiency state in vivo. Theresultant protein synthesis would be, in onesense, comparable to a cure as observed in theuncommon hemophilic patient that has receivedliver transplantation. A distinction with organtransplantation is that in gene therapy, onesown cells are transduced (the process of addinga functional gene to a cell). Thus immuno-suppression, which is necessary to preventrejection of the transplanted organ, and itsrelated toxicity, is not necessary. Initial studiesled to considerable optimism that hemophiliacould be cured [1]. The basic elements of genetransfer, including different vectors, deliverysystems, and target cells used with factors VIIIor IX, have been reviewed [2, 3, 4].

A distinction should be drawn between genetherapy to modify a hosts somatic cells and otherhighly experimental approaches that lead totransgenic animals. Such animals (e.g., Dolly)are capable of passing on the geneticmodification to the next generation as it involvesall of their cells, including those of their germline(eggs and sperm). Transgenic sheep were createdto synthesize human factor IX and contained thehuman factor IX DNA in all tissues [5]. Thisapproach may be useful in generating female

animals such as pigs that secrete human factorsVIII or IX in their milk [6, 7].

A second distinction is between gene transferand gene repair. All of the human and most ofthe preclinical animal studies have focused ongene transfer, where extra copies of the normalcoding DNA (cDNAs) for factors VIII or IX areinserted into cells that were previously unable tosynthesize a normal clotting factor. For gene


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2 Gene Therapy for the Hemophilias

repair, relatively limited success has beenrealized to date in either cultured cells or animalmodels, although there are some promisingnovel approaches being developed that willlikely be worthy of trials in animals.

Animal models of hemophilia gene transfer with

viral-derived vectors have been the most reliableat providing sustained therapeutic levels of theclotting factors. These animal models includemutations that have occurred naturally in dogs(then bred to form hemophilic colonies) or inmice made factor VIII- or IX-deficient bytargeted gene disruption, so-called geneknockout mice [8].

Vectors for Gene Transfer

The transfer of DNA into cells for gene therapyis accomplished by transduction, a controlledprocess that is mediated by vectors or vehiclesthat attach to a cell surface and facilitate entryinto the cell. This is in contrast to transfection,where cell membranes are disrupted and DNAinsertion occurs by physical or electrochemicalmeans. Vectors for transduction are commonlyderived from viral nucleic acid backbones, asthese are more efficient than current non-viralpreparations. The portion of the virus thatdirects cell attachment and entry is retained.Vector systems with promising preclinical

animal studies relevant to gene therapy for thehemophilias are summarized in Table 1. The

final section of this review highlights

considerations for their use in clinical trials.

Retroviral vectorsAn early strategy in gene therapy was to useretroviral vectors (gamma retroviruses or onco-retroviruses) to transduce dividing cells in tissue

culture and return the transduced cells to thehost animal by transplantation [9]. However,transgene expression levels in a variety of celltypes were considerably higher in culture thanin vivo secondary to gene silencing aftertransplantation. These problems led to efforts toimprove methods to administer vectors directlyto animals and transduce the cells in vivo. Theprinciple of long-term (sustained) expressionfollowing in vivo administration of a retroviralvector was established when hemophilia B dogsexpressed normal canine factor IX for over two

years, although levels were insufficient tocorrect the bleeding tendency. Furthermore,because of the retroviral vectors requirementfor dividing cells, these dogs underwent partialhepatectomy in order to increase the number ofcells in division at the time of portal veininfusion of the viral construct.

Other strategies used to generate sustained invivo factor IX expression from retroviral vectorsinclude gene transfer into neonatal hosts or intothe livers of adult animals stimulated by growth

factors to induce hepatocyte proliferation.

Table 1:Viral Vectors Used for Preclinical Gene Transfer of Factors VIII or IX

Vector NucleicAcid

Advantages Limitations Reference

Retroviral RNA efficient transduction

genomic integration

persistent expression

oncogene derivation

random insertion

cell division-dependent

(except lentiviral)

Van Damme et al, 2004

Adenoviral DNA transduces non-dividing cells

accommodates largecDNAs

high level ofexpression

immune responses to AV

episomal (no integration)

transient expression

Thorrez et al, 2004

Adeno-AssociatedViral

DNA integration (partial)

persistent expression

different serotypes

limited size of cDNA

possible rearrangements

Couto, 2004


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vector carrying the canine factor IX cDNA todogs with severe hemophilia B, either bymultiple intramuscular injections or infused intothe portal vein, expression of therapeutic levelsof factor IX for longer than five yearshas beenobtained. A canine B domain-deleted factor VIIIcDNA has also been inserted into an AAV

vector and portal vein injection of this constructhas resulted in expression of therapeutic levelsoffactor VIII in hemophilic dogs and mice [15,16]. Again, expression of the factor VIIItransgene in dogs has been maintained for morethan 5 years.

Non-viral vectorsThe advantage of a non-viral delivery system isto avoid potential toxicities of viral vectors, be itfrom integration or inflammatory and/orimmune responses to viral capsid proteins. As

non-integrating systems, however, one would notanticipate prolonged expression. Furthermore, amajor difficulty is stabilizing the DNA andefficiently inserting it into cells in vivo [17]. Therehas been surprisingly long-term expression offactors IX and VIII, however, in immuno-deficient hemophilic mice, suggesting thatepisomal expression can persist providing onehas a highly efficient vector construct. In factorVIII-deficient normal mice, however,alloimmunization occurred even when a speciesspecific murine factor VIII was used [18]. The

degree to which the immune response isinfluenced by initially high levels of geneexpression and/or cytokine responses evokedduring delivery remains to be assessed. A novelalternative approach is to use non-viral vectorscontaining transposons (a piece of DNA that canmove from one location in the gene and reinsertat another site) derived from ancestral geneticsequences. Vectors with transposons canintegrate into host cell genomic DNA if deliveredconcomitantly with the transposase enzyme.Recently, it was demonstrated that a transposonvector containing the factor IX cDNA was able to

integrate into mouse liver cells and direct long-term gene expression and partial phenotypiccorrection of hemophilia B mice [19].

Alternative cell typesThe hepatocyte is well established as the site ofsynthesis of factor IX and the liver isthe primaryorgan of synthesis of factor VIII. There may wellbe contributions from both hepatocytes andsinusoidal endothelial cells for the latter. Blood

outgrowth endothelial cells can be harvested,transduced in vitro with factor VIII and serve as asource for gene transfer [20, 21]. Hematopoieticcells have long been a target due to theaccessibility of stem cells in peripheral blood.Although yields have been disappointingly low,there is early success with factor VIII using a

lentiviral vector [22]. Megakaryocyte expressionof factor VIII would have a potential advantageof co-expression of the stabilizing von Willebrandfactor protein. This has been accomplished incultured cells using a megakaryocyte-specificpromoter [23] and has shown very promising invivo results, even in the face of high levels ofFVIII inhibitors [24].

Gene repairRNA/DNA hybrid oligonucleotides have beenused to correct point mutations [25]. Although

limited success has been reported in someanimal systems, efficiency, particularly in vivo, isa major problem with this approach. There issome evidence that nonsense mutations can betransiently suppressed at the ribosomal levelusing aminoglycoside antibiotics such asgentamycin. This could potentially convertsevere hemophilia to a moderately severephenotype in selected patientswith this type ofhemophilic mutation. Use in 5 hemophilicsubjects with nonsense genotypes, however,failed to show a clinically significant effect [26].

Trans-splicing is another mechanism for generepair at the RNA level, and could potentially beapplicable to inversions; early studies withfactor VIII in mice suggest that it may be afeasible approach for the hemophilias [27] and,if successful, would avoid the potential toxicitiesof viral vector gene transfer. Optimal deliverysystems for the latter remain to be developed.

Human Trials of Hemophilia GeneTransfer

As vector systems capable of delivering the factorIX or VIII cDNAs to host cells have beenoptimized to express sufficient quantities of theclotting proteins in vivo, an important issue hasbeen the design of therapeutic trials in humanpatients. Issues of safety are paramount inassessing the potential risks versus the possibilityof making hemophilia less severe or enacting along-term cure. In the following sections, theresults of initial human clinical trials of


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Gene Therapy for the Hemophilias 5

hemophilia gene therapy will be summarized.Potential safety concerns are discussed for eachvector system, followed by considerations as towhich patients represent appropriate candidatesfor these initial human studies.

To date, five clinical trials of hemophilia gene

therapy have been completed involving a totalof 41 patients (Table 2). All of these studies havebeen phase I/II trials in which the primarypurpose has been to assess potential toxicity oftreatment (phase I) and to determine if there areany early signs of therapeutic benefit (phase II).It should be noted that no patient showed anyevidence of inhibitor development as a responseto gene transfer. None of the patients treated inthese trials experienced significant long-termadverse effects but two individuals hadtransient side effects related to their treatment

with viral vectors.

Hemophilia AThere have been three trials of factor VIII genetherapy given to 26 subjects with hemophilia A.Two of these used viral vectors delivered in vivo,whereas a third used non-viral delivery tocultured, autologous cells [28].

In the non-viral vector system, autologousfibroblasts were harvested from a skin biopsyand during culture were transfected by

electroporation with the cDNA of a B domain-deleted factor VIII gene containing a fibronectinpromoter to limit the potential for expression inother cell types [28]. B domain-deleted factorVIII protein is commercially available asharvested from cultured, transfected cells andappears comparable in recovery, survival, andclinical effect for treatment or prevention ofbleeding to full-length (wild-type; B domain-included) factor VIII. The fibroblasts from thesesubjects were then selected for their ability toexpress factor VIII and secrete it into the culturemedia. These cells were then implanted

laparoscopically into the peritoneal cavity byinjection into omental fat pads. Patients weregiven factor VIII concentrate to prevent bleedingassociated with the invasive procedure. Onlyminor adverse events were encountered, suchthat the procedure appears quite safe and nolong-term complications occurred. Post-therapy,clotting factor levels were measured and, insome, were at or slightly above the 1% limit ofdetection for the clotting activity assay. Within

several days to a few weeks, however, all levelsreturned to pre-treatment baselines. Anotherpotential benefit seen in some of these subjectswas a reduction of spontaneous bleedingepisodes that required exogenous infusions offactor VIII concentrates. However, it has to berecognized that this latter outcome may be

difficult to distinguish from a placebo effect.

Another factor VIII gene transfer trial used anearly generation retroviral vector derived from amurine leukemia virus [29]. The vectorcontained the viral promoter sequence toexpress a B domain-deleted factor VIII. It wasadministered by simple intravenous infusionson three successive days. The vector infusionsand subsequent courses were well toleratedwith no complications noted. Post-therapyclotting factor VIII clotting activities, although

somewhat variable as in the fibroblast study,showed no consistent elevations above the 1%limit of detection. Furthermore, there was nodose-response seen. Within several days to afew weeks, all activities were at pre-treatmentlevels. However, again some subjects reportedfewer spontaneous bleeding episodes in theweeks following gene transfer. Although datawere limited, there may have been a prolongedsurvival of transfused factor VIII that wouldsuggest a low (albeit undetectable by routineassays) level of transgenic factor VIII circulating.

An adenoviral vector that was helperdependent, that is, stripped of its viral genes,and with the full-length factor VIII cDNAinserted, was administered intravenously to onepatient [30]. It included an albumin promoter tolimit expression primarily to the hepatocytes.Although some factor VIII clotting activitiessuggested a possible effect that was independentof infused factor VIII required to treat sporadicroutine bleeding episodes, there was nodefinitive, sustained response and levels foundwere near the level of detection so may have

been spurious. The patient experienced atransient fall in his platelet count and showedtransient laboratory evidence of liver celldamage, presumably as a toxic effect of thecapsid or coating protein of the transducingvector system.


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Table 2:Clinical Trials of Gene Transfer for Hemophilia A and B

Vector/promoter(study sponsor)

Delivery/primary organ Subjects(N)

Results

Hemophilia A:

Non-viral/fibronectinwith B domain-deleted factor VIIIcDNA (TranskaryoticTherapies)

omental implantation after exvivo electroporation, selection& growth of autologous,cultured fibroblasts

12 no sustained responses

possible transient, factor VIII levels insome but at level of detection

possible decreased infusionrequirements in some

nevbilaw

Retroviral/viral-LTR with B domain-deleted factor VIIIcDNA (Chiron)

3 daily intravenousdoses/liver


possible prolonged survival oftransfused factor VIII in some


n

Adenoviral/albuminwith full-length factorVIII cDNA (GenStar)

intravenous/liver 1 no sustained response

possible transient factor VIII levels butat level of detection

trdth

Hemophilia B:

Adeno-associatedviral, AAV-2/CMVwith factor IX cDNAand partial intron(Avigen)

intramuscular/multipleinjections into vastus lateralisbilaterally


persistent vector & expressiondemonstrated up to 10 mo in musclebiopsies

nevtoin

Adeno-associatedviral, AAV-2/ alpha-antitrypsin with factorIX cDNA (Avigen)

intra-hepatic via catheterwith transient balloonocclusion of flow to hepaticartery

7 no sustained responses at highest dose, one patient achieved

transient factor IX level of 12%


trdptobl


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Hemophilia BTwo trials have been concluded using adeno-associated viral vector constructs in 15 subjectswith hemophilia B [31, 32]. Although factor IX isgenerally expressed at higher levels than factorVIII, it is smaller than albumin and thus has alarger volume of distribution. Furthermore, on a

molar basis, it circulates in a 300-fold excessover factor VIII.

The first trial targeted gene transfer into musclecells in severe hemophilia B subjects, and thoughlong-term gene expression was demonstrated,systemic factor IX levels were not significantlyincreased. In the second trial, the liver was thetarget organ. At the highest dose, transientefficacy was demonstrated though two subjectsexperienced delayed liver toxicity associated withthis vector.

The muscle trial used an adeno-associatedviral vector with a viral promoter expressing afactor IX minigene that contained the factor IXcDNA with a partial intron sequence [33]. Asdoses escalated, multiple injection sites into thelateral thigh were performed. Muscle biopsies,performed at 2 and up to 10 months, showedpersistent vector and, by immunofluorescence,factor IX protein expression. The onlycomplications of note were related to the biopsysites. Of note, no inhibitors developed; semen

samples were screened and were negative forviral sequences, excluding significant germlinetransmission. Results of the efficacy, however,were disappointing compared to the doseresponses observed in preclinical studies,including those in hemophilic dogs. This mayrelate to a relatively low density of cell surfacereceptors for adeno-associated virus in themuscle group selected for human studies, theserotype used, and the fact that myocytes and

myotubes have a limited ability to -carboxylatefactor IX. Thus more injection sites would beneeded as opposed to larger amounts of vectorinjected per site.

In the liver trial, the adeno-associated viralvector was engineered for a higher level ofexpression and a hepatocellular-specificpromoter was used [34, 32]. The vector wasdelivered through a catheter fluoroscopicallyplaced into the hepatic artery as a radiologicprocedure. A balloon was inflated totemporarily stop the hepatic circulation, as used

for localized infusion of chemotherapy formalignancies. The portal circulation preventedliver ischemia. Efficient hepatic gene transferwas achieved as one of two patients treated atthe highest vector dose had factor IX clottingactivities that reached 12%, clearly a significantincrement due to the gene transfer. However,

success was only temporary as activity fell tobaseline by four weeks, by which time thepatient showed transient laboratory evidence ofliver cell toxicity. An additional patient, enteredat a reduced vector dose, showed slightchemical liver dysfunction that may also reflectan immune response to the incoming vectorcapsid proteins [35]. This complication led to thediscontinuation of this trial [36]. Currently, twonew AAV factor IX trials are in the final stagesof planning and will likely start to enrollpatients during 2008.

Human Safety Considerations withViral Vectors

Retroviral vectorsRetroviral vectors are packaged in producercells by viral gene products that are supplied intrans by helper constructs. Precautions areneeded to ensure that replication-competentvirus is not produced through recombination ofhelper and vector genes. Insertional mutagenesisis another safety concern because sites of

retroviral vector integration cannot becontrolled. Since the vector carries its ownpromoter that can activate any nearby genes,integration in proximity to a proto-oncogenecould predispose that cell and its progeny todevelop a neoplastic growth pattern, i.e., cancer.Considering the size of the entire genome, this isa very unlikely complication even if integrationis not entirely random. There is considerableevidence in patients who have received olderforms of retroviral vectors with a variety ofdifferent cDNAs that these retroviral vectors are

safe and well tolerated, at least short term.However, the development of T-cell leukemia intwo patients treated in a recentimmunodeficiency gene therapy trial suggeststhat this type of complication can occur incertain, probably rare, instances, dependingupon the type of gene being delivered, and theselection of cells for delivery [37]. Clearly non-viral vectors would be preferable to avoid anyrisk of replication-competent virus generation or


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insertional mutagenesis. However, non-viralvectors are much less efficient at entering cellssuch that delivery and stability must beimproved considerably before they can beconsidered as potentially therapeutic.

Adenoviral vectors

Adenoviral vectors are safe in the sense thatthey do not integrate. Several copies cantransduce the same cell and the copy numberneeds to be kept low enough to prevent cellulartoxicity and cell death. As a cause of thecommon cold, it is possible that someindividuals will have sufficiently highantibody titers against the parent type ofadenoviral vector to prevent cell entry.Adenoviral vectors also activate the early, innatephase of the host immune system that, in itsmost extreme form, can result in an

overwhelming systemic inflammatory state anddeath [38]. To avoid the later, adaptive immuneresponses to adenoviral proteins,gutless/helper-dependent vectors areparticularly appealing. However, their ability topersist for sufficiently long periods to providesustained clotting factor production remains tobe demonstrated, and they will require furthermodifications to allow repeated transductions.A study in dogs with hemophilia A suggeststhat this may become possible [13]. A variety ofstrategies to limit the immune responses to

adenoviral vectors are being investigated [39].

Adeno-associated viral vectorsAdeno-associated viral vectors persist forextended periods of time in host cell nuclei butonly a small proportion of the vector integratesinto the genome. The small amount of thevector that integrates raises the same concernsof oncogene activation that apply withretroviral vectors. This could be problematic inhepatic gene therapy to an individual whocarries hepatitis C, as that virus is alreadycausing increased hepatocyte turnover.Administration into muscles avoids thehepatitis C risk although this route is known tobe more immunogenic.

Although only one of the few hemophilia Bdogs injected intramuscularly with caninefactor IX cDNA-AAV vector developed atransient anti-factor IX antibody response [40],the incidence could be higher in humanpatients. The original dog colony used has a

single missense mutation with undetectableplasma factor IX antigen, although the presenceof a messenger RNA factor IX transcriptsuggests that traces of a factor IX protein maybe synthesized, at least intracellularly, and thismay decrease the overall risk of an anti-factorIX antibody developing. Gross gene alterations

and nonsense mutations are more commonlyassociated with alloimmunization in bothhemophilia A and B.

There was no evidence of alloimmunization inthe eight previously transfused severehemophilia B patients (all with missensegenotypes) receiving AAV intramuscularly[33]. The alloimmunization rate in hemophiliaA following concentrate therapy is at least anorder of magnitude greater than inhemophilia B. However, due to its larger size

and intravascular distribution, factor VIIIdelivered by an intramuscular route wouldlikely have limited access to the circulation. Aninitial concern with AAV liver delivery was thetransient appearance of vector in human semen[34]. The presence in sperm cells, however, hasbeen essentially excluded. Furthermore, in amurine model, the AAV vector failed totransduce sperm cells, even when incubateddirectly with sperm at very high levels [41].Thus germline transmission, althoughtheoretically possible for viral vectors, does not

appear to occur.

In the one individual who achieved the firstunequivocal response to AVV-factor IXdelivered to the liver, there was evidence of adelayed immune response and transient hepatictoxicity [34, 32, 35]. The degree to which thisrepresents a serotype issue with the vectorand/or a host response not observed inpreclinical studies remains to be determined.

Patient participationSelectionof study subjects requires carefulconsideration, especially in a bleeding tendencythat can usually be controlled by infusiontherapy [42]. It is critical to ensure that potentialsubjects are counselled as to possible risks.Complications encountered in clinical trials todate need to be presented in an unbiasedmanner. Furthermore, the potential subjectsmotivation must be carefully evaluated foraltruism to advance the science, understandingthat there is little to no chance of personal gain,


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especially in phase I/II clinical trials. Protocolsmust ensure that potential subjects are notsimply trying to please the investigator,especially where s/he is also that individualstreating physician.

It has generally been agreed that initial clinical

studies should involve consenting adulthemophilic patients. Whether or not HIV-positive individuals should be eligible is adebatable issue. If so, certain anti-retroviralmedications may interfere with retroviralvectors. On the other hand there are a number ofhemophilic patients who are long-termsurvivors of HIV infection, some of whom haveremained healthy, even without proteaseinhibitor therapy, for over 20 years. Anyinclusion of HIV-positive adults should requirethat they are currently asymptomatic, have low

viral loads, and reasonable CD4 lymphocytecounts.

An additional concern is the potential forgermline transduction. Although this is veryunlikely for most of the vectors being proposedfor clinical trials, it would seem prudent to selectpatients initially that will not be having childrenor are willing to use a barrier form ofcontraception for a period of time around thevector administration. Careful follow-upscreening, such as with PCR amplification, to

look for even traces of vector DNA in spermsamples, for example, should help settle theissue of whether or not germline integration iseven a slight risk.

In the United States, the Food and DrugAdministration (FDA) needs to review thepreclinical data and approve each protocol forgene therapy before the vector can beconsidered as an investigational new drug (IND)and clinical experimentation can proceed.Furthermore, a national recombinant advisoryboard committee (RAC) of the National

Institutes of Health (NIH) provides additionalguidance and oversight for gene therapyprotocols. Once the qualifications for inclusionin a study are determined and protocols arefinalized and approved nationally, furtherconsideration and approval of the protocols andconsent forms is required by institutional reviewboards (IRBs) for human subjects of eachparticipating institution. Rigorous safety

standards are required to ensure safe,reproducible manufacture of the viral vectors.

Even though there are perhaps slight andpossibly unforeseen risks of gene therapy forhemophilia, animal model data suggests it canbe effective and safe. Its safety to date in other

patient groups strongly supports the carefuldesign of clinical studies to determine the abilityof gene therapy to lessen the severity of thebleeding tendency, and possibly to cure, factorIX or VIII deficiencies in patients withhemophilia B and A, respectively.

Conclusion

Hemophilia continues to represent a leadingcandidate condition for the successfulapplication of gene therapy. Over the pastdecade, dramatic advances have been made inthe preclinical assessment of hemophilia genetransfer and there are now many reports of long-term success in hemophilic mice and somesuccess, lasting for several years, in hemophilicdogs. More recently, the initial human trials ofhemophilia gene therapy have not shown anypersistent adverse effects, but increments ofclotting factor levels have been at best minimaland short-lived. It seems likely that for long-term therapeutic levels of clotting factor to beachieved in humans, several remaining

challenges will need to be overcome includingthe avoidance of host immune responses to thevector and the transgene product, increasedtransduction efficiencies, and the developmentof transgene constructs that maintain persistentexpression following administration. However,despite these challenges, there is still reason foroptimism, and the development of safe, effectivemethods to provide a cure for hemophiliaremains feasible.

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17. Gomez-Vargas A, Hortelano G. Nonviralgene therapy approaches to hemophilia. SemThrombos Hemostas 2004; 30: 197-204.

18. Ye P, ThompsonAR, Sarkar R, Lillicrap DP,Kaufman RJ, Ochs HD, Rawlings DJ, MiaoCH. Transgene-specific, species-independent immune response against

factor VIII after naked DNA transfer.MolTher2004; 10: 117-126.

19. Mikkelsen JG, Yant SR, Meuse L, Huang Z,Xu H, Kay MA. Helper-independent SleepingBeauty transposon-transposase vectors forefficient nonviral gene delivery andpersistent gene expression in vivo.Mol Ther2003; 8: 654-665.

20. Lin Y, Chang LM, Solovey A, Healey JF,Lollar P, Hebbel RP. Use of blood outgrowthendothelial cells for gene therapy for

hemophilia A. Blood 2002; 99: 457-462.21. Matsui H, Shibata M, Brown B, Labelle A,

Hegadorn C, Andrews C, Hebbel RP,Galipeau J, Hough C, Lillicrap D. Ex vivogene therapy for hemophilia A thatenhances safe delivery and sustained in vivofactor VIII expression from lentivirallyengineered endothelial progenitors. StemCells 2007; 25(10):2660-9.


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22. Tiede A, Eder M, vonDepka M, Battmer K,Luther S, Kiem HP, Ganser A, Scherr M.Recombinant factor VIII expression inhematopoietic cells following lentiviraltransduction. Gene Ther2003; 10: 1917-1925.

23. Shi Q, Wilcox DA, Fahs SA, Kroner PA,

Montgomery RR. Expression of humanfactor VIII under control of the platelet-specific alpha IIb promoter inmegakaryocytic cell line as well as storagetogether with VWF.Mol Genetics Metabol2003; 79: 25-33.

24. Shi Q, Wilcox DA, Fahs SA, Weiler H, WellsCW, Cooley BC, Desai D, Morateck PA,Gorski J, Montgomery RR. Factor VIIIectopically targeted to platelets istherapeutic in hemophilia A with high-titerinhibitory antibodies.J Clin Invest. 2006;

116(7):1974-82.25. Kren BT, Bandyopadhyay P, Steer CJ. In

vivo site-directed mutagenesis of the factorIX gene by chimeric RNA/DNAoligonucleotides. Nature Med 1998; 4: 285-290.

26.James PD, Rivard GE, Poon MC, Warner M,Raut S, McKenna S, Leggo J, Lillicrap DP.Aminoglycoside suppression of nonsensemutations in severe hemophilia. Blood 2003;102: 53a (abstract #176).

27. Chao HJ, Mansfield SG, Bartel RC,Hiriyanna S, Mitchell LG, Garcia-Blanco M,Walsh CE. Phenotype correction ofhemophilia A mice by spliceosome-mediated RNA trans-splicing. Nature Med2003; 9: 1015-1019.

28. Roth DA, Tawa NE Jr, O'Brien JM, TrecoDA, Selden RF. Nonviral transfer of the geneencoding coagulation factor VIII in patientswith severe hemophilia A. N Engl J Med2001; 344: 1735-1742.

29. Powell JS, Ragni MV, White GC, Lusher JM,Hillman-Wiseman C, Moon TE, Cole V,Ramanathan-Girish S, Roehl H, Sajjadi N,

Jolly DJ, Hurst D. Phase 1 trial of FVIII genetransfer for severe hemophilia A using aretroviral construct administered byperipheral intravenous infusion. Blood 2003;102: 2038-2045.

30. White GC. Clinical study of a gutlessadenovirus vector (MAXADFVIII) fortreatment of hemophilia A. Presented at theNational Hemophilia FoundationsWorkshop on Gene Therapy for theHemophilias, La Jolla, CA, April, 2003.

31. High KA. Clinical gene transfer studies forhemophilia B. Sem Thrombos Hemostas 2004;30: 257-267.

32. Manno CS, Pierce GF, Arruda VR, Glader B,Ragni M, Rasko JJ et al. Successfultransduction of liver in hemophilia by AAV-Factor IX and limitations imposed by thehost immune response. Nat Med. 2006;12(3):342-7.

33. Manno CS, Chew AJ, Hutchison S, LarsonPJ, Herzog RW, Arruda VR et al. AAV-mediated factor IX gene transfer to skeletal

muscle in patients with severe hemophilia B.Blood 2003; 101: 2963-2972.

34. High K, Manno C, Sabatino D, Hutchison S,Dake M, Razavi M et al. Immune responsesto AAV and to factor IX in a phase I study ofAAV-mediated, liver-directed gene transferfor hemophilia B.Mol Ther2004; 9:abstract#1002.

35. Mingozzi F, Maus MV, Hui DJ, Sabatino DE,Murphy SL, Rasko JE et al. CD8(+) T-cellresponses to adeno-associated virus capsid

in humans. Nat Med. 2007; 13(4):419-22.

36. Kaiser J. Gene therapy. Side effects sidelinehemophilia trial. Science 2004; 304: 1423-1425.

37. Williams DA, Baum C. Gene therapy: newchallenges ahead. Science 2003; 302: 400-401.

38. Raper SE, Chirmule N, Lee FS, Wivel NA,Bagg A, Gao GP, Wilson JM, Batshaw ML.Fatal systemic inflammatory responsesyndrome in a ornithine transcarbamylasedeficient patient following adenoviral gene

transfer.Mol Genet Metab 2003; 80: 148-155.

39. Schagen FHE, Ossevoort M, Toes REM,Hoeben RC. Immune responses againstadenoviral vectors and their transgeneproducts: a review of strategies for evasion.Crit Rev Oncol Hematol 2004; 50: 51-70.

40. Herzog RW, Dobrzynski E. Immuneimplications of gene therapy for hemophilia.Sem Thrombos Hemostas 2004; 30: 215-226.


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41. Couto L, Parker A, Gordon JW. Directexposure of mouse spermatozoa to very highconcentrations of a serotype-2 adeno-associated virus gene therapy vector fails tolead to germ cell transduction. Hum GeneTher2004; 15: 287-291.

42. Ragni MV. Hemophilia gene transfer:comparison with conventional proteinreplacement therapy. Sem ThrombosHemostas 2004; 30: 239-247.

General References

1. Mamman EF. Preface: Gene therapy inhemophilia A and B in animals and humans:Current status and perspectives. SemThrombos Hemostas 2004; 30: 157-159.

The issue, edited by VandenDriessche T &Chuah MKL, contains several reviews citedbelow that discuss much of the olderprimary literature on vector developmentand cellular and preclinical animal genetransfer.

2. Pierce GF, Lillicrap D, Pipe SW,Vandendriessche T. Gene therapy,bioengineered clotting factors and noveltechnologies for hemophilia treatment.JThromb Haemost. 2007; 5: 901-6.

3. Hough C, Lillicrap D.Gene therapy forhemophilia: an imperative to succeed.JThromb Haemost 2005; 3: 1195-205.


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