292 final results of phase i trial of GMCSF gene TGFß2 antisense gene autologous tumor cell (TAG) vaccine in advanced cancer

292 Final Results of Phase I Trial of GMCSF Gene TGFß2 Antisense Gene Autologous Tumor Cell (TAG) Vaccine in Advanced Cancer Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The America[.]

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CLINICAL GENE & CELL THERAPY ORAL ABSTRACT SESSION

288 CASPALLO: Phase I Clinical Trial of

Allodepleted T Cells Transduced with Inducible

Caspase 9 Suicide Gene after Haploidentical Stem

Cell Transplantation

Antonio Di Stasi,1 Siok K Tey,1 Yuriko Fujita,1 Russell Cruz,1

Caridad Martinez,1 Alana Kennedy-Nasser,1 George Carrum,1

Carlos A Ramos,1 Stephen M Gottschalk,1 Karin Straathof,1 Lin

Yu-Feng,1 Enli Liu,1 April Durett,1 Bambi Grilley,1 Hao Liu,1

Barbara Savoldo,1 Eric Yvon,1 Teresita Lopez,1 Oumar Diouf,1

Deborah Lyon,1 Adrian Gee,1 Ellen Vitetta,2 John Schindler,2

Catherine M Bollard,1 Robert A Krance,1 David M Spencer,1

Gianpietro Dotti,1 Cliona M Rooney,1 Helen E Heslop,1 Malcolm

K Brenner.1

1 Baylor College of Medicine, Texas Children’s Hospital and

Methodist Hospital, Houston, TX; 2 UT Southwestern Medical

School, Dallas.

Adoptive T cell transfer after haploidentical CD34+ stem cell

transplantation (haplo- CD34+ SCT) is often complicated by graft

versus host disease (GVHD) We have previously demonstrated that

addback of allodepleted T cells accelerates T cell recovery and

anti-viral immune-reconstitution after haplo-CD34+ SCT Since disease

relapse remains high we have developed a protocol to increase the

number of allodepleted T cells administered whilst using a suicide

gene to destroy the infused cells should GVHD occur Phase I-II

studies, employing Herpes Simplex Virus thymidine kinase (HSVtk)

suicide gene modi ed donor T cells, demonstrated that donor T cells

can be eliminated in allograft recipients after gancyclovir (GCV)

administration Potential drawbacks include: immunogenicity of

HSVtk, killing restricted to dividing cells, unintended elimination

of donor T cells in case of GCV administration and reports of

GCV resistance We developed an alternative suicide gene strategy

based on an inducible human caspase 9 molecule (iCasp9) that can

be activated by administering a chemical inducer of dimerization

(AP1903) leading to apoptosis of transduced cells Our system is

likely not immunogenic, killing is not restricted to dividing cells

and the transgene does not preclude GCV administration Brie y,

donor peripheral blood mononuclear cells were co-cultured with

recipient irradiated EBV-transformed lymphoblastoid cells (40:1) for

72 hrs, allodepleted with a CD25 immunotoxin and then transduced

with a retroviral supernatant carrying the iCasp9 suicide gene and

a selection marker (∆CD19); ∆CD19 allowed enrichment to 〉90%

purity via immunomagnetic selection Three patients have received

iCasp9+ T cells after haplo-CD34+ SCT, at dose levels between

1x106 to 3x106 cells/kg Infused T cells could be detected in vivo

by  ow cytometry (CD3+∆CD19+) or qPCR as early as day 7 after

infusion, with a maximum fold expansion of 170±5 (day 29±9 after

infusion) Two patients developed grade I/II aGVHD and AP1903

administration caused 〉90% ablation of CD3+∆CD19+ cells, within

30 minutes of infusion, with resolution of skin and liver aGvHD

within 24hrs, showing that iCasp9 transgene is functional in vivo

Ex vivo experiments con rmed this data Furthermore, the residual

allodepleted T cells were able to expand and were reactive to viruses

(CMV) and fungi (Aspergillus fumigatus) (IFN-γ production)

In conclusion, addback of iCasp9+ allodepleted T cells after haplo

CD34+ SCT allows a signi cant expansion of functional donor

lymphocytes in vivo and a rapid clearance of alloreactive T cells

with resolution of aGvHD

Clinical Gene & Cell Therapy Oral Abstract Session

289 Therapeutic DNA Vaccination Followed by Standard-of-Care Therapy in Patients with Chronic

Hepatitis C: A Rapid Clearance of Viremia

Matti Sallberg,1 Lars Frelin,1 Helmut M Diepolder,2 Maria C

Jung,3 Iacob Mathiesen,4 Michael Fons,5 Gustaf Ahlen,1 Margaret Chen,1 Ola Weiland.1

1 Laboratory Medicine and Medicine, Karolinska Institutet, Stockholm, Sweden; 2 Medicine, Ludvig-Maximilian University,

Munich, Sweden; 3 Immusystems GmbH, Munich, Germany; 4 Otivio

AS, Oslo, Norway; 5 Inovio Biomedical, San Diego, CA.

The hepatitis C virus (HCV) effectively establishes chronic infections A strong T cell response to HCV proteins can prevent chronic infections, whereas the HCV-speci c T cell response is severely impaired during a chronic HCV infection We recently concluded the worlds  rst phase I/II clinical study of a therapeutic DNA vaccine delivered by in vivo electroporation against an infectious disease, in 12 patients with chronic HCV genotype 1 infection We could show that the treatment was well tolerated and that HCV-speci c T cell responses lasting four weeks or more were induced four out of nine patients in the two highest dose groups In the same four patients, and one more from the highest dose group, transient reductions in the viral replication of 0,6 to 2,4log10 were observed This provides a proof-of concept for the ability of

DNA-based therapeutic vaccination to induce immune responses that can transiently control HCV replication All study patients are currently commencing therapy with standard-of-care (SOC), consisting of ribavirin and interferon-alpha (IFN) Importantly, of the  rst four patients, who at start of SOC had a viral load ranging from 293,000

to 1,700.00 copies/mL, three had a rapid viral response (RVR) with negativity for HCV RNA by a quantitive PCR (<50 copies/mL) already at treatment weeks three (n=1) and four (n=2) The fourth patient was HCV RNA negative by treatment week 12 suggesting

a complete early viral response (cEVR) Such a rapid loss of HCV RNA is highly unusual for patients infected with HCV of gentype 1

Thus, therapeutic vaccination delivered by in vivo electroporation has transient antiviral effects as a monotherapy, but may be of an immediate clinical bene t in combination with SOC therapy in

patients with chronic HCV of genotype 1

290 Sustained Alpha-Sarcoglycan Gene Expression in LGMD2D Following Gene Transfer

Jerry R Mendell,1 Louise R Rodino-Klapac,1 Xiomara Rosales-Quintero,1 Brian D Coley,1 Gloria Galloway,1 Sarah Lewis,1 Vinod

Malik,1 Christopher J Shilling,1 Barry J Byrne,3 Thomas Conlon,3

Katherine J Campbell,4 William G Bremer,4 Christopher M

Walker,4 Zarife Sahenk,1 K Reed Clark.1

1 Center for Gene Therapy, Research Institute at Nationwide Children’s Hospital, Columbus, OH; 2 Pediatrics, University of

Florida College of Medicine, Gainesville, FL; 3 Center for Vaccines and Immunology, The Research Institute at Nationwide Children’s

Hospital, Columbus, OH.

Limb-girdle muscular dystrophy (LGMD) type 2D is de ned as a de ciency of a-sarcoglycan (α-SG), a sarcolemmal transmembrane protein contributing to membrane stability There are few if any treatment options for this disease We previously reported a successful trial of SGCA gene transfer in three patients with persistence of gene expression for at least three months The number of α -SG positive

 bers reached 57%, 69%, and 62% and western blot showed a 4 to 5 fold increase in each subject with the maximum restoration of α-SG in any block 76% of normal control muscle Persistent gene expression was complemented with restoration of the full sarcoglycan complex and an increase in mean  ber diameter in one subject analyzed

at 3 months To evaluate whether long term gene expression was achievable, a second cohort of 3 patients was evaluated at 6 months

As in the  rst cohort, a double-blind, randomized controlled trial was initiated in LGMD2D subjects using rAAV1 with full length human SGCA under control of a truncated muscle creatine kinase (tMCK) promoter The extensor digitorum brevis (EDB) muscle received 3.25 X 1011 vector genomes on one side, while the control side was given saline All analyses were done prior to breaking the blind

The entire EDB muscle was removed from both sides at 6 months

in all three subjects Transgene speci c PCR analysis demonstrated expression on only one side in each subject which corresponded with gene expression Subject 1 increased α -SG by 2 fold on the side of gene transfer reaching wild-type levels This was accompanied by an increase in mean muscle  ber diameter, 28.2 ± 11.1 to 52.2 ± 13.1

Subject 2 demonstrated robust gene expression, again equivalent to wild-type levels on the side of gene transfer MHCI and CD4, CD8 mononuclear cells were upregulated on the side of gene transfer in subjects 1 and 2 ELISpots for antigen speci c production of IFN-γ secretion were monitored beginning pre-gene transfer Subject 1 had

a transient minimal response to AAV1 capsid pools at days 14 and

28 Patient 2 showed no response to α-SG or AAV1 capsid peptide pools In contrast to the  rst two patients, Subject 3 had pre-existing immunity to AAV1 (neutralizing antibodies) and an ELISpot IFN-γ response within the  rst week to AAV following gene transfer suggesting a memory response Levels of gene expression were also reduced in this subject compared to others Overall the promising results from this study with gene expression persisting for 6 months lays the foundation for potentially achieving long-term sustained correction of the underlying gene defect in muscular dystrophy

However, the in uence of pre-existing immunity to AAV may affect initial muscle  ber transduction or sustained gene expression

291 Advanced Generation RGD-Modi ed Oncolytic Adenovirus ICOVIR-7 in the Treatment of Cancer Refractory to Other Modalities

Petri Nokisalmi,1,2 Sari Pesonen,1,2 Sophie Escutenaire,1,2 Mari Raki,1,2 Vincenzo Cerullo,1,2 Ramon Alemany,3 Juanjo Rojas,3

Maria Rajecki,1,2 Lotta Kangasniemi,4 Anna Kanerva,1,5 Timo Joensuu,6 Laura Ahtiainen,1,2 Akseli Hemminki.1,2

1 Cancer Gene Therapy Group, Transplantation laboratory &

Haartman Institute & Finnish Institute for Molecular Medicine, University of Helsinki, Helsinki, Finland; 2 HUSLAB, Helsinki University Central Hospital, Helsinki, Finland; 3 Translational Research Laboratory, IDIBELL-Institut Català d’Oncologia, L’Hospitalet de Llobregat, Barcelona, Spain; 4 Oncos Therapeutics Inc., Helsinki, Finland; 5 Department of Obstetrics and

Gynecology, Helsinki University Central Hospital, Helsinki, Finland; 6 International Comprehensive Cancer Center Docrates, Helsinki, Finland.

We treated 21 cancer patients with a single round of oncolytic adenovirus ICOVIR-7 with doses ranged from 2 x 1010 VP to 1 x 1012

VP All patients had advanced and metastatic solid tumors refractory

to standard therapies ICOVIR-7 features an RGD-4C modi cation

of the  ber HI-loop of serotype 5 adenovirus for enhanced entry into tumor cells Tumor selectivity is mediated by an insulator, a modi ed E2F promoter and an Rb binding site deletion, while replication

is optimized with E2F binding hairpins and a Kozak sequence

ICOVIR-7 treatment was well tolerated with mild to moderate fever, fatigue, elevated liver transaminases, chills and hyponatremia One patient had grade 3 anemia but no other serious side effects were seen At baseline, 8/20 of evaluable patients had neutralizing antibody titers (NAb) against the ICOVIR-7 capsid Treatment resulted in NAb induction within four weeks in 16/17 patients No elevations

of serum pro-in ammatory cytokines were detected Viral genomes were detected in the circulation in 18/21 of patients after injection

and 7/15 of samples were positive several weeks later suggesting virus replication Overall, objective evidence of anti-tumor activity was seen in 9/17 evaluable patients Clinical bene t in radiological analysis was seen in 5/12 evaluable patients, consisting of one partial response, two minor responses and two cases of stable disease In addition, 3/3 evaluable patients had a decrease in tumor density In summary, ICOVIR-7 treatment seems safe, results in anti-cancer activity and is therefore promising for further clinical testing

292 Final Results of Phase I Trial of GMCSF Gene-TGFȕ2 Antisense Gene Autologous Tumor Cell (TAG) Vaccine in Advanced Cancer

John Nemunaitis,1,2,3,4,5 Padmasini Kumar,5 Neil Senzer,1,2,3,4,5

Joseph Kuhn,6 Minal Barve,1 Jairo Olivares,1 Gerald Edelman,1

Cynthia Bedell,1 Yang Yu,5 Mitchell Magee,7 Beena O Pappen,5

Gladice Wallraven,5 Alex W Tong,5 Phillip B Maples.5

1 Mary Crowley Cancer Research Centers, Dallas, TX; 2 Medical City HCA, Dallas, TX; 3 Baylor Sammons Cancer Center, Dallas, TX; 4 Texas Oncology, P.A., Dallas, TX; 5 Gradalis, Inc., Dallas, TX;

6 General and Oncology Surgery Associates, Dallas, TX; 7 Cardio Thoracic Surgery Associates of North Texas, Dallas, TX.

Both GMCSF gene transduced vaccine and TGFβ2 antisense gene vaccine, in separate trials, have demonstrated bene cial effects without evidence of toxicity in advanced cancer patients The GMCSF transgene directly stimulates increased expression of tumor antigen(s) and enhances dendritic cell migration to the vaccination site TGFβ2 blockade following intracellular TGFβ2 antisense gene expression reduces production of immune inhibiting activity at the vaccine site We recently completed a phase I clinical investigation of the combined GMCSF/TGFβ2 antisense TAG vaccine under BB IND

13650 Nineteen patients [6/13, M/F; median age 58 yrs; median number prior chemotherapy regimens 2; cancer: melanoma (3), lung (2), colon (4), breast (2), neuroendocrine (4), other (4)] have received between 1 and 12 vaccinations Harvested malignant tissue was processed to a single cell suspension, and cells were transfected via electroporation with our novel TAG expression vector and irradiated (median number vaccines/patient, 10) Patients received monthly intradermal injections in two dosing cohorts (1x107 cells/ injection or 2.5x107 cells/injection) for a minimum of 4 months No grade 3, 4 toxic events related to therapy were observed in the 19 treated patients One patient was unevaluable for response due to discontinuation after one month related to travel dif culty Sixteen

of 18 patients maintained stable disease for 3 months Two patients maintained stable disease for one year; a third (melanoma) achieved complete response (CR) and remains in CR now 18 months after  rst

vaccination (Figure 1).Figure 1

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Conclusion: GMP manufacturing of TAG vaccine is feasible, safety

is achieved and evidence of clinical bene t is demonstrated

293 Administration of Tumor-Speci c Cytotoxic

T Lymphocytes Engineered to Resist TGF-ß to

Subjects with EBV-Associated Lymphomas

Catherine M Bollard,1 Gianpietro Dotti,1 Stephen Gottschalk,1 Enli

Liu,1 Andrea M Sheehan,2 George Carrum,1 Hao Liu,1 Chung-Che

Chang,3 Adrian P Gee,1 Malcolm K Brenner,1 Helen E Heslop,1

Cliona M Rooney.1

1 Center for Cell and Gene Therapy, Baylor College of Medicine,

Houston, TX; 2 Department of Pathology, Texas Children’s

Hospital, Houston, TX; 3 The Methodist Hospital and TMHRI, Weill

Medical College of Cornell University, Houston, TX.

EBV-associated Hodgkin’s Lymphoma (HL) and some

non-Hodgkin’s lymphomas (NHL) express EBV proteins which are

potential targets for immunotherapy Lymphomas arising in the

immune-competent host, however, utilize multiple immune evasion

strategies to evade host T cell responses These include limited

expression of EBV antigens, so that only the subimmunodominant

EBNA1 and LMP1 and 2 antigens are expressed (type II latency); and

production of immunosuppressive cytokines, such as TGF-ß We  rst

tested our hypothesis that cytotoxic T lymphocytes (CTL) enriched

for speci city to LMP antigens would have ef cacy in patients

with EBV+ lymphoma LMP-CTL were generated from 29 patients

with EBV+ HL and NHL using antigen-presenting cells genetically

modi ed to overexpress either LMP2 alone (n=16), or LMP2 and

inactive LMP1 (∆LMP1) (n=14) No immediate toxicity was observed

following infusion 14/15 high-risk and/or multiply relapsed patients

who received LMP-CTL as adjuvant treatment remain in remission

for a median of 2.5 years (range 6 months to >5 years) Of the 14

patients who had detectable disease at the time of CTL infusion, 11

had clinical responses (78%), including 8 (57%) complete responses

The median duration of the clinical responses is 1.5 years However,

36% of patients were either resistant to CTL therapy (n=3) or relapsed/

progressed >9 months after CTL therapy (n=2), suggesting the

importance of additional tumor immune escape mechanisms TGF-ß

inhibits the growth and function of effector T cells and is secreted

both by lymphoma cells and by in ltrating T regulatory cells To

test our hypothesis that the infused CTL can be made resistant to the

TGF-ß produced by these tumors, LMP-CTL from 2 patients with

relapsed EBV+ve HL one of whom had failed initial CTL therapy,

were transduced with a retrovirus vector encoding the

dominant-negative TGF-ß type II receptor (DNR) Unlike non-transduced CTL,

DNR-transduced CTL were resistant to the anti-proliferative effects of recombinant TGF-ß in vitro Both patients received 4 x 107CTLs/m2

without toxicity Neither patient received additional therapy following these CTL infusions DNR-transduced T-cells have been detected by quantitative real-time PCR ampli cation in the peripheral blood of both patients for >3 and >6 months respectively post infusion One patient had a complete remission and the other has a mixed clinical response Hence, immunotherapy with CTL targeting LMP antigens

is well tolerated in patients with EBV+ lymphoma and can induce durable clinical responses Furthermore, for patients resistant to CTL therapy, the use of DNR-transduced CTL may be bene cial

294 Ef cient Engraftment of MGMTP140K Gene-Modi ed CD34+ Cells Following Nonmyeloablative BCNU Conditioning in Patients with Glioblastoma

Jennifer E Adair,1 Brian C Beard,1 Alexander Spence,2 Jason Rockhill,2 Daniel L Silbergeld,2 Robert Rostomily,2 Pamela Becker,2 Marc Chamberlain,2 Maciej Mrugala,2 Hans-Peter Kiem.1,2

1 Fred Hutchinson Cancer Research Center, Seattle, WA;

2 University of Washington, Seattle, WA.

Glioblastoma, the most common primary malignant brain tumor in adults, confers poor prognosis, with a median survival

of 15 months, notwithstanding aggressive treatment Furthermore, patients whose tumors present with an unmethylated methylguanine methyltransferase (MGMT) promoter display a poorer prognosis than patients with methylated MGMT promoter In an effort to improve therapy and outcome for patients with unmethylated MGMT, and thus high wild type MGMT expression, chemotherapy combining alkylating agents carmustine (BCNU) or temozolomide (TMZ) with the wild type MGMT active site inhibitor O6-benzylguanine (O6BG) has been used, but has been associated with dose limiting hematopoietic toxicity We have previously demonstrated in the dog and nonhuman primate model ef cient gene marking, in vivo selection and chemoprotection of hematopoietic cells from O6BG and BCNU

or TMZ following transplantation with MGMTP140K gene-modi ed CD34+ cells Based on these  ndings, we have developed a clinical gammaretroviral vector produced by Phoenix-GALV packaging cells for use in a Phase I/II clinical trial This study (National Clinical Trials Registry: NCT00669669) enrolled three glioblastoma patients following surgical resection, diagnosis and pathological subtyping

of unmethylated tumor MGMT promoter status in each patient

Enrolled patients underwent standard radiation therapy, followed

by G-CSF mobilization, apheresis and conditioning with 600 mg/

m2 BCNU prior to infusion of gene-modi ed cells Gene marking

in pre-infusion colony forming units (CFUs) was 70.6%, 79.0% and 74.0% in patients 1, 2 and 3 respectively, as determined by CFU-PCR

The dose of BCNU resulted in patient neutrophil counts below 500/

µL for only 2, 3 and 1 days, respectively, in the three patients, and

no platelet count below 28,000/µL Following engraftment, initial gene marking in peripheral blood mononuclear cells (PBMNCs) and sorted granulocytes ranged between 0.37 to 0.84 and 0.33 to 0.83 provirus copies, respectively, by real-time PCR Intracellular MGMT-staining of PBMNCs confirmed MGMT expression

Furthermore, gene marking in CFUs from CD34-selected cells at 1-2 months post-transplant ranged from 28.5% to 47.4% Initial integration site analysis in Patients 1 and 2 has demonstrated that multiple gene-modi ed clones are contributing to hematopoiesis and

is in process for Patient 3 All three patients have been treated with at least one cycle of O6BG and TMZ post-transplant with preliminary evidence of in vivo selection of gene-modi ed cells No additional extra-hematopoietic toxicity has been observed in any patient to date

We believe that these data demonstrate the feasibility of achieving signi cant engraftment of MGMTP140K- modi ed cells with a

RNA VIRUS VECTORS

tolerated dose of BCNU Further follow-up will determine whether this approach will allow for dose escalation of temozolomide and/or result in clinical bene t

295 Thymic Renewal and Anti-Leukemic Effect

in Adults after Haplodentical Transplantation and Suicide Gene Therapy

Luca Vago,1 Giacomo Oliveira,1 Maddalena Noviello,1 Corrado Soldati,1 Domenico Ghio,1 Roberto Nicoletti,1 Immacolata Brigida,1 Alessandro Aiuti,1 Maria Teresa Lupo Stanghellini,1

Jacopo Peccatori,1 Attilio Bondanza,1 Katharina Fleischhauer,1

Claudio Bordignon,2 Fabio Ciceri,1 Chiara Bonini.1

1 San Raffaele Scienti c Institute, Milano, Italy; 2 MolMed SpA, Milan, Italy.

Introduction: Hematopoietic Stem Cell Transplantation (HSCT) from partially-HLA matched (haploidentical) family donors represents

a promising therapy for high-risk leukemia, but requires appropriate strategies to control the adverse reactions mediated by the partially incompatible transplanted immune system In a recent phase II clinical trial (TK007 study) we demonstrated that the infusion of donor lymphocytes transduced with the Herpes Simplex Virus Thymidine kinase (HSV-Tk) suicide gene allows to control Graft-versus-Host Disease and to rapidly provide an effective and polyclonal anti-infective T cell repertoire (Ciceri and Bonini et al., Lancet Oncology, 2009) Here we investigated the role of the infused HSV-Tk+ cells in Graft-versus-Leukemia effect and in promoting thymic renewal after transplantation Methods: Twenty-eight adult patients received Tk+

donor T cells in the context of the TK007 study In a selected subset

of patients, post-transplantation thymic function was assessed after validating the methods in healthy pediatric and adult controls Single joint T cell Receptor Excision Circles (sjTREC) were quanti ed by qPCR, and the proportion of CD31+ recent thymic emigrants (RTEs)

in CD4+ nạve T cells was measured with immunophenotype analysis

Thymic output was correlated with thymic volume, assessed by CT scans Alloreactivity against leukemic blasts was studied by mixed lymphocyte cultures Results: Post-transplant recovery of Tk- Nạve

T cells occurred, reaching values of healthy controls in approximately one year At the moment of T cell immune reconstitution (de ned

as CD3+ cells > 100/µl peripheral blood), the CD4+ nạve T cell subset was almost entirely comprised by CD31+ RTEs, and this percentage remained higher than in age-matched controls also in subsequent months Comparison between RTE frequency before and after HSV-Tk+ cell add-backs suggested a direct role of the infused cells in promoting thymopoiesis Accordingly, CT scans documented

an increase in thymic volume following HSV-Tk+ cell add-backs

Besides promoting thymic renewal, HSV-Tk+ cells displayed also a direct antitumor effect, as demonstrated by their selective enrichment and functional activity against leukemic blasts Consistent with their

ex vivo alloreactivity, in two of the treated patients HSV-Tk+ cells drove in vivo selection of immunoresistant leukemic variants with genomic loss of the mismatched HLA haplotype Conclusions: These data show that the infusion of suicide gene-modi ed T cells prompts the renewal of thymic activity, which contributes to the recovery of

a polyclonal T cell repertoire Contextually, the infused transduced cells mediate also a direct antitumor effect, through their recognition

of allogeneic determinants on leukemic cells Ef cacy of HSV-Tk+

cells in the context of haploidentical HSCT for leukemia is currently being assessed in a phase III clinical trial

296 Update on a Clinical Gene Therapy Trial for Adenosine Deaminase De cient Severe Combined Immune De ciency (ADA-SCID)

Kit L Shaw,1 Yeong “Christopher” Choi,1 Linda Muul,2

Robert Sokolic,2 Denise A Carbonaro,1 Alan Ikeda,1 Monika Smogorzewska,3 Jayashree Jagadeesh,2 Elizabeth Garabedian,2

Ami Shah,3 Neena Kapoor,3 Michael S Hersh eld,4 Fabio Candotti,2 Donald B Kohn.1

1 Microbiology, Immunology, and Molecular Genetics and Pediatrics, UCLA, Los Angeles, CA; 2 National Human Genome Research Institute, National Institutes of Health, Bethesda, MD;

3 Research Immunology/Bone Marrow Transplant, Childrens Hospital Los Angeles, Los Angeles, CA; 4 Biochemistry, Duke University, Durham, NC.

We report results on a clinical trial of gene therapy for ADA-de cient SCID Between 2001 and 2002, four ADA-ADA-de cient SCID patients were treated by retroviral-mediated ADA cDNA transfer

to their bone marrow CD34+ cells using two slightly different vectors (MND-ADA and GCsap-M-ADA) They were not given pre-transplant cytoreductive chemotherapy and remained on PEG-ADA throughout Only short term (months) low level (<0.01%) gene marking was seen in peripheral blood cells of the two older subjects (15 and 20 years old at time of treatment), whereas low level but persistent gene marking continues in the two younger subjects (4 and

5 years old at time of treatment) now for seven years Between 2006 and 2009, six additional ADA-de cient patients were treated using the same gene transfer protocol, but they were taken off PEG-ADA enzyme replacement therapy and given 75-90 mg/m2 busulfan prior

to cell reinfusion One subject had unrecognized pre-existing trisomy

8 mosaicism, failed to reconstitute, and underwent a successful matched unrelated donor BMT Two subjects developed infections requiring hospitalization and were restarted on PEG-ADA ERT The other 3 subjects remain well off PEG-ADA (3 years, 2.5 years, and 15 months post-procedure), have gene marking in peripheral blood cells

in the range of 10%, with ADA enzyme expression in PBMC near or into the normal range We have also treated 3 subjects on a Phase II protocol, which uses only the MND-ADA vector A 15-year old male

is 6 months out from the procedure His lymphocyte count has not yet recovered and PBMC ADA enzyme activity, although detectable,

is low A child, who was 4 ½ months old at the time of treatment, is

4 months out from the procedure and already has 1/3 normal ADA enzyme activity in her PBMCs The third child, an 8-year old, is less than 2 months out from the procedure and is too early to evaluate at this time These  ndings demonstrate that combining pre-transplant myelo-reductive conditioning and withdrawing PEG-ADA with gene therapy for ADA-de cient SCID can be bene cial

RNA Virus Vectors

297 Sensitive In Vivo Models To Assess the Risk of Insertional Mutagenesis in the Liver upon Vector Systemic Delivery

Marco Ranzani,1,2 Daniela Cesana,1,2 Manfred Schmidt,3 Francesca Sanvito,4 Fabrizio Benedicenti,1 Cynthia Bartholomä,3 Maurilio Ponzoni,4 Alessio Cantore,1,2 Lucia Sergi Sergi,1 Claudio Doglioni,4

Christof VonKalle,3 Luigi Naldini,1,2 Eugenio Montini.1

1 San Raffaele Telethon Istitute for Gene Therapy, Milan, Italy;

2 San Raffaele University, Milan, Italy; 3 National Center for Tumor Diseases, Heidelberg, Germany; 4 Pathology, San Raffaele Scienti c Institute, Milan, Italy.

Ef cient liver gene transfer and long term transgene expression may allow the treatment of several hepatic and systemic diseases However, vector integration may occasionally lead to transformation

of hepatocytes, as reported for AAV in mice Therefore, sensitive

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