Download RNA Virus Vectors

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; 2MolMed 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 antiinfective 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+ naïve 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- Naïve
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+ naïve 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; 2National Human Genome
Research Institute, National Institutes of Health, Bethesda, MD;
3
Research Immunology/Bone Marrow Transplant, Childrens
Hospital Los Angeles, Los Angeles, CA; 4Biochemistry, Duke
University, Durham, NC.
We report results on a clinical trial of gene therapy for ADAdecient SCID. Between 2001 and 2002, four 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 PEGADA 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; 3National Center for
Tumor Diseases, Heidelberg, Germany; 4Pathology, 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
Molecular Therapy Volume 18, Supplement 1, May 2010
Copyright © The American Society of Gene & Cell Therapy
S113