1163 corrective gene transfer into bone marrow CD34+ cells for adenosine deaminase (ADA) deficiency: results in four patients after one year of follow up

1163 Corrective Gene Transfer into Bone Marrow CD34+ Cells for Adenosine Deaminase (ADA) Deficiency Results in Four Patients after One Year of Follow Up Molecular Therapy �������� ��� ���� �����������[.]

Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts

Copyright ®The American Society of Gene Therapy

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ADVANCES IN CLINICAL GENE THERAPY

increase in dye uptake in the tumors during therapy Tumor biopsies

from two of these patients revealed that the dye uptake was due to

heavy immune infiltration and not tumor progression The third

patient refused all further tumor therapy but showed a decrease in

dye uptake following an increase in his decadron dose, suggesting

that the increased dye uptake in this patient was also due to immune

infiltration In these situations, debulking the inflamed tumor may

benefit the patient and prolong survival Overall, post treatment

tumor biopsies in the four patients who were tested showed increased

levels of CD3+, CD4+, CD8+, CD45RO+, and CD68+ effector cell

infiltrates compared to the primary tumor Based upon these findings,

we have initiated a Phase II clinical trial of a TGF-β

antisense-modified allogeneic tumor cell vaccine in non-small cell lung cancer

and are preparing for a Phase II clinical trial in colorectal carcinoma

and a Phase II/III clinical trial in glioma

Authors Shawler, Chu, and Fakhrai are employees of and hold

stock options for NovaRx Corporation, the sponsor of the clinical

trial

1162 Correction of the Immune and Metabolic

Defect of ADA-SCID by Stem Cell Gene Therapy

Combined with Nonmyeloablative Conditioning

Alessandro Aiuti,1 Lucia Gaetaniello,1 Federica Cattaneo,1

Francesca Ficara,1 Grazia Andolfi,1 Barbara Cassani,1 Antonella

Tabucchi,2 Filippo Carlucci,2 Roberto Miniero,3 Memet Aker,4

Shimon Slavin,4 Maria Grazia Roncarolo§,1 Claudio Bordignon§.1

1 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET),

Milan, Italy; 2 Dip MISEMB, Sez di Biochimica Clinica, Univ of

Siena, Siena, Italy; 3 Univ of Turin, Turin, Italy; 4 Dep of BM

Transplantation and Pediatrics, Hadassah Univ Hospital,

Jerusalem, Israel.

Adenosine deaminase (ADA)-deficient SCID was the first

inherited disease treated with gene therapy In this disorder, the

purine metabolic defect leads to impaired immune functions,

recurrent infections, and non-lymphoid abnormalities We recently

described an improved clinical protocol based on gene transfer into

autologous bone marrow (BM) hematopoietic stem cells (HSC),

associated with nonmyeloablative conditioning to favor multilineage

engraftment Four patients have been enrolled in this gene therapy

trial All patients lacked an HLA-identical sibling donor, and enzyme

replacement therapy was not administered because it was either not

available or was discontinued due to side effects Patients were

treated with busulfan (2 mg/Kg/day at day -3 and -2), prior to

reinfusion of transduced BM CD34+ cells The procedure was well

tolerated and hematopoiesis recovered normally after a transient

myelosuppression The duration of neutropenia and

thrombocytopenia varied between patients, with Pt3 experiencing

the longest period of myelosuppression The patients have a follow

up of 29, 22, 9 and 3.5 months, respectively Engraftment of gene

corrected HSC, with differentiation into multiple lineages, was

demonstrated in all four patients The highest levels of engraftment

were observed in Pt1 and Pt3, in whom the ADA transgene was

detected in virtually all T, B, and NK cells, and in a significant

proportion of myeloid, erythroid and megakaryocytic cells (3-12%)

The extended follow up in Pt1 confirmed the normalization of

peripheral blood (PB) lymphocyte counts (2500/μl at last control)

with full correction of the T-cell defect Serum immunoglobulin

levels were normalized and specific antibodies were produced at

protective titers after vaccination or exposure to infective agents In

Pt2, who received one log less transduced CD34+ cells than Pt1, the

degree of immune reconstitution was less pronounced (PB

lymphocyte counts: 300/μl) Nevertheless, all circulating T cells

contained the ADA transgene, displayed a polyclonal TCRVβ

repertoire, and were able to proliferate normally in vitro in response

to mitogens In Pt3, PB lymphocyte counts raised to 1000/μl after

gene therapy, with normal lymphoid development Furthermore, T-cell proliferative responses normalized and serum immunoglobulin levels increased Biochemical studies showed a dramatic decrease in RBC dAXP metabolites in all patients, associated with ADA expression in multiple lineages and amelioration of the systemic toxicity A systematic analysis of vector integration sites by inverse-PCR in PB and BM subpopulations is in progress A polyclonal pattern of vector integrations was found in PB T cells of Pt1 and Pt3, whereas fewer integration sites were detected in Pt2 The treated children are presently at home, clinically well, and did not experience any adverse effect or severe infections, in the absence of enzyme replacement therapy, and with the longest follow up at 126 weeks

§M.G.R and C.B equally contributed to the work

1163 Corrective Gene Transfer into Bone Marrow CD34+ Cells for Adenosine Deaminase (ADA) Deficiency: Results in Four Patients after One Year of Follow-Up

Fabio Candotti,1 Greg Podsakoff,2 Shepherd H Schurman,1 Linda

M Muul,1 Barbara C Engel,2 Denise A Carbonaro,2 G

Jayashree Jagadeesh,1 Peiman Hematti,1 Laura M Tuschong,1 Charley S Carter,1 W Jay Ramsey,1 Christopher Choi,2 Monika Smogorzewska,2 Michael S Hershfield,3 Elizabeth J Read,1 John

F Tisdale,1 Cynthia E Dunbar,1 Donald B Kohn.2

1 National Institutes of Health, Bethesda, MD, United States;

2 Children Hospital Los Angeles, Los Angeles, CA, United States;

3 Duke University Medical Center, Durham, NC, United States.

Mutations affecting expression and/or function of adenosine deaminase (ADA) result in a rare form of immunodeficiency that affects both T and B cell function ADA deficiency can be cured with allogeneic bone marow transplantation, which supported the development of corrective gene transfer into hematopoietic stem/ progenitor cells as an alternative form of therapy ADA deficient patients lacking suitable donors can also be treated with enzyme replacement therapy through intramuscular injections of pegylated ADA (PEG-ADA) Previous trials of ADA gene transfer into CD34+ cord blood and bone marrow cells have failed to achieve significant marking in patients concomitantly treated with PEG-ADA Low gene transfer and insufficient gene expression in vivo combined with the negative effects of PEG-ADA on the survival advantage of gene-corrected cells were identified among possible causes of these negative results In August 2001, we began a clinical trial evaluating the efficacy of two new retroviral vectors expressing high levels of ADA The MPSV and MND LTRs were used in these vectors (GCsapM-ADA and MND-ADA, respectively) in combination with modern transduction conditions (SCF, MDGF, Flt3-L, CH-296 fibronectin fragment) to target bone marrow CD34+ cells of four ADA-deficient patients on PEG-ADA treatment Except for the first patient who received only cells transduced with GCsapM-ADA, isolated CD34+ cells were divided in two fractions, separately transduced with one of the vectors and re-infused into the patients who continued PEG-ADA treatment Transduction efficiency of final cell products ranged between 1.4%-48% and 0.8%-66% for GCsapM-ADA and MND-ADA, respectively Infused cells expressed 7-235 U (nmol/108 cells/min) of ADA activity (from 0.9-3.5 U in untransduced cells, normal: 100-150 U) To date, patients have been monitored for 12-15 months after infusion No serious adverse events were observed GCsapM-ADA-marked cells (1:104 -1:105) became undetectable in 3 out of 4 patients by 4-8 months after infusion and were intermittently present for up to one year in one patient who carries LAM-PCR evidence of policlonal integrations without clonal dominance Higher levels of marking with MND-ADA (up to ~5:10² in one patient) were observed in all the 3 patients who received this vector MND-ADA marked cells have persisted at levels ~1:105 up to the most recent determinations

Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts

ADVANCES IN CLINICAL GENE THERAPY

and were generally more prevalent among lymphoid than myeloid

cells No clear changes of patients’ immunological profiles have

been observed to date Although low transduction efficiency in 2

patients (~1-11%) and possible reduced thymopoiesis in the older

subjects (15 and 20 years old) can in part account for our results, the

concurrent administration of PEG-ADA is likely to have played a

role by reducing the advantage and consequent expansion of

gene-corrected cells in vivo

1164 A Clinical Attempt To Treat JAK3-Deficient

SCID Using Retroviral-Mediated Gene Transfer to

Bone Marrow CD34+ Cells

Brian P Sorrentino,1 Taihe Lu,1 James Ihle,1 Rebecca H Buckley,2

John M Cunningham.1

1 Hematology/Oncology, St Jude Children’s Research Hospital,

Memphis, TN, United States; 2 Pediatrics, Duke University

Medical Center, Durham, NC, United States.

JAK3 deficiency is a rare autosomal recessive form of severe

combined immunodeficiency (SCID), accounting for about 7% of all

SCID cases Murine models of JAK3 deficiency have been developed

and have shown that insertion of a retroviral vector expressing a

normal copy of the JAK3 gene into hematopoietic stem cells can

correct the immunodeficient phenotype, both in irradiated animals

and in unconditioned recipients Based on these results, we have

developed a clinical gene therapy approach for this disorder and

have treated a single patient with JAK3-deficient SCID An

MSCV-based vector expressing the human JAK3 cDNA was generating in

PG13 packaging cells, and a clinically certified high titer clone was

derived Transduction of EBV-immortalized B cells from the

JAK3-deficient patient showed high levels of JAK3 protein expression A

2 ½ year old boy with JAK3 null SCID was treated with this vector,

after having previously failed two allogeneic transplant attempts

using his mother as a donor Bone marrow cells were harvested and

transduced on 2 occasions; the first done using a Nexell CD34

purification column failed due to an insufficient number of cells

after transduction The second CD34+ purification was done with

the Miltenyi Clinimacs system and yielded 2.4 X 106 CD34+ cells/

kg for transplant, with 85% of the pre-transplant CFU-C positive

for vector DNA sequences by PCR These cells were infused without

conditioning and the patient has now been followed for 7 months

Vector copy number determinations have shown stable levels of

marked cells for 200 days, at levels corresponding to 1 copy per

10,000 cells Vector mRNA transcripts have also been detectable by

PCR, and when corrected for the estimated proportion of transduced

cells, are expressed at relatively high levels, equivalent to that seen

in the producer cells Despite this demonstration of vector gene

transfer and expression, there has been no evidence of immune

reconstitution to date This single patient experience suggests that

other variables may be important in determining the response to

gene therapy, including the age of the patient at the time of treatment,

and the degree of thymic competence In particular, the ability of

the thymic remnant to mature T cells could be defective in this

patient and explain the lack of response both to the gene therapy

attempt and the previous allogeneic transplant attempts

1165 Results of Phase I Clinical Trial of Plasmid-Dystrophin Intramuscular Administration into Duchenne and Becker Dystrophy Patients

Norma B Romero,1 Olivier Benveniste,2 Aurore Choquel,2 Christine Payan,1 Glenn E Morris,3 Jean-Gerard Guillet,4 Jean-Claude Kaplan,4 France Leturcq,4 Serge Braun,5 Brigitte Mourot,5 Christine Thioudellet,5 Patrick Squiban,5 Serge Herson,2 Michel Fardeau.1

1 Institut de Myologie, Groupe Hospitalier Pitié-Salpêtrière, Paris, France; 2 Clinical Medicine, Groupe Hospitalier Pitié-Salpêtrière, Paris, France; 3 North East Wales Institute, Wrexham, United Kingdom; 4 ICGM - INSERM U445, and Groupe Hospitalier Cochin Port-Royal, Paris, France; 5 Transgene S.A., Strasbourg, France.

Duchenne dystrophy is a recessive X-linked inherited disorder with deletions or mutations in the dystrophin gene Clinical onset occurs usually before the age of four years and is fatal (death during the third decade of life) A milder form (Becker dystrophy) is of more variable phenotype, but with abnormalities in the same

dystrophin gene After extensive pre-clinical studies in mdx mice

and GRMD dogs we have carried out the first gene therapy phase I clinical trial in both Duchenne and Becker patients The goal of this study was to provide indications on safety of a full-length human dystrophin-plasmid administration and exogenous dystrophin expression in DMD/BMD patients Many ethical aspects were considered, including age and sequential inclusion of the patients, and low plasmid dosage Complete data of each patient was carefully analysed by a steering committee composed of investigators, promoters and external, independent experts, before enrolment of the following patient Three cohorts of 3 patients, presenting with large deletions, were injected in the radial muscle with either a single injection of 200 μg (cohort 1) and 600 μg (cohort 2) of plasmid, or

2 injections 2 weeks apart with 600 μg plasmid (cohort 3) In all patients, a muscle biopsy was performed in the injected site 3 weeks after the first injection Each biopsy was serially sectioned and studied for the presence of plasmid (PCR) and for dystrophin expression (nested RT-PCR + immunohistochemistry) The histological aspect of the muscle biopsy and local inflammatory processes were analysed Besides the usual biochemical / cytological blood parameters (including muscle enzymes), patients were also followed for specific immunological endpoints (DNA and anti-dystrophin cellular and immunological responses) for 3 months after plasmid injection Normal dystrophin expression was found in few muscle fibers of 2/3 patients of the first cohort and in 1/3 patients of the second cohort All the 3 patients of the 3rd cohort displayed significant amounts of weakly dystrophin-stained muscle fibers Plasmid was detected in the injected muscle sample in all patients All patients showed perfect tolerance to the plasmid administration Neither anti-DNA nor anti-dystrophin immune response was found

As demonstrated by MRI, EMG and muscle strength analysis, the procedure did not impair the injected muscle function

These results show for the first time that exogenous dystrophin

expression can be obtained in DMD/BMD skeletal muscle in vivo

following gene transfer, and without adverse effects This very cautious approach paves the way for further developments We are currently working extensively (in collaboration with JA Wolff and Mirus Corp.), on a more ambitious, intra-arterial delivery administration of human full-length dystrophin plasmid, that we intend to evaluate in a next human clinical trial