605 The Infusion of Suicide Gene Modified Donor T Cells after Hematopoietic Stem Cell Transplantation Prompts Thymic Renewal in Adult Patients by an IL 7 Dependent Mechanism Molecular Therapy Volume 1[.]
Trang 1Molecular Therapy Volume 19, Supplement 1, May 2011
Copyright © The American Society of Gene & Cell Therapy S231
CLINICAL GENE & CELL THERAPY ORAL ABSTRACT SESSION
for the Treatment of Parkinson Disease: Phase I
Clinical Trial Update
Kyriacos Mitrophanous,1 Bechir Jarraya,2,3 Scott Ralph,1 Helene
Lepetit,2 James Miskin,1 Jean-Marc Gurruchaga,2 Gilles Fenelon,2
Sabrina Boulet,1 Caroline Jan,2 Gilles Bonvento,2 Pierre Brugiere,2
Susan Kingsman,1 Stuart Naylor,1 Philippe Hantraye,2,3 Philippe
Remy,2 Stephane Palfi 2,3
1 Oxford BioMedica (UK) Ltd, Oxford, United Kingdom; 2 Henri
Mondor Hospital, Paris, France; 3 CEA-CNRS MirCen, Fontenay
aux Roses, France.
Parkinson’s disease (PD) is a debilitating neurodegenerative
condition that results from the destruction of dopamine-producing
(dopaminergic) neurons in the substantia nigra The loss of dopamine,
a key neurotransmitter involved in coordinating motor control,
results in movement abnormalities such as bradykinesia (slowness of
movement), rigidity and postural instability L-Dopa and dopamine
agonists provide the primary standard of care for PD and are highly
effi cacious in the early stages of disease However their long term
use is associated with severe motor side effects that seriously impact
on the quality of life These side effects are believed to be caused by
the fl uctuating nature of dopaminergic stimulation that arises from
oral drug administration We have developed a lentiviral vector
(ProSavin®) derived from the equine infectious anaemia virus
expressing the three key dopamine biosynthetic enzymes (tyrosine
hydroxylase, aromatic L-amino acid decarboxylase and GTP
cyclohydrolase-1), with the aim of providing a continous source of
dopamine in the striatum ProSavin was previously demonstrated to
mediate dopamine production and behavioural correction in rat and
non-human primate models of PD A phase I, open label clinical study
has been initiated in which nine PD patients have received ProSavin
in three cohorts in the dose escalation part of the trial All the patients
have completed at least 3 months follow up and the fi rst cohort are
now approaching their thrid year post treatment ProSavin has been
demonstrated to be safe and well tolerated at both doses There were
no “OFF” state dyskinesias, no immune responses to ProSavin and
no serious adverse events Furthermore, encouraging signs of effi cacy
have been observed on a number of endpoints including UDRS Part
IIII, patient diary and quality of life measures An update on the trial
and future plans for ProSavin® will be presented
Wiskott-Aldrich Syndrome
Samantha Scaramuzza,1 Francesca Ferrua,2 Maria Carmina
Castiello,1 Luca Biasco,1 Marita Bosticardo,1 Costanza Evangelio,2
Maria Pia Cicalese,2 Miriam Casiraghi,2 Anna Ripamonti,1 Stefania
Giannelli,1 Monica Salomoni,3 Andrea Finocchi,4 Alessandra
Biffi ,1,2 Fabio Ciceri,5 Anna Villa,1,6 Maria Grazia Roncarolo,1,7
Luigi Naldini,1,7 Alessandro Aiuti.1,4
1 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET),
Milan, Italy; 2 Pediatric Immunohematology and BMT Unit,
Scientifi c Institute HS Raffaele, Milan, Italy; 3 MolMed Spa,
Milan, Italy; 4 Univ of Rome “Tor Vergata”, Rome, Italy; 5 Div of
Hematology, Scientifi c Institute HS Raffaele, Milan, Italy; 6
IRGB-CNR, Cagliari, Italy; 7 Università Vita Salute, San Raffaele, Milan,
Italy.
Wiskott-Aldrich Syndrome (WAS) is an X-linked immunodefi ciency
characterized by thrombocytopenia, autoimmunity and lymphomas
Gene therapy with ex vivo transduced autologous hematopoietic
stem cells (HSC) could represent a valid therapeutic alternative
to allogenic HSC transplant We previously demonstrated that a
lentiviral vector (LVV) encoding for human WAS under the control
of an homologous 1.6 kb promoter effi ciently corrected human and
mouse cells We set up and validated a transduction protocol for CD34+ cells derived from bone marrow (BM) or mobilized peripheral blood (MPB) using a clinical grade purifi ed LVV We obtained robust transduction of patients’ progenitor cells, expressing WASp after differentiation to megakaryocytes, without evidence of toxicity Studies in immunodefi cient mice showed that human transduced CD34+ cells were able to normally engraft and differentiate towards lymphoid and myeloid cells Analyses of vector integrations showed
polyclonal integration patterns in vitro and in engrafted cells in vivo;
vector mobilization to host cells and germline transmission of the LVV were excluded by several molecular assays On the basis of these results and preclinical studies in the WAS-KO mouse, a phase I/II gene therapy protocol was opened in April 2010 Patients will receive preconditioning with anti-CD20 mAb and reduced intensity busulfan and fl udarabin; ATG will be included in case of autoimmune manifestations The fi rst enrolled patient was a 5.7 year old boy lacking an HLA-compatible donor He was treated with autologous
BM and MPB derived transduced CD34+ cells, showing a vector copy number (VCN) of 1.4 and 1.9 respectively, with high gene transfer effi ciency (88-92% in clonogenic progenitors) The patient did not experience toxicity, recovered well from transient neutropenia and
is currently well, independent from platelet transfusions, 6 months after gene therapy Engraftment analyses revealed the presence of transduced cells at substantial levels in peripheral blood granulocytes and monocytes (VCN: 0.3-0.4) and at higher levels in T, B and NK lymphocytes (VCN range 0.5-1.0) as expected from the selective advantage Engraftment was present in multiple lineages of the bone marrow (VCN range 0.2-0.9), including CD34+ cells and clonogenic progenitors (25% transd.) WASp expression was detected in peripheral blood monocytes, platelets and all lymphocyte lineages Long-term studies will provide key information on the safety and effi cacy of gene therapy for WAS patients using LVV transduced HSC in combination with reduced intensity conditioning
Donor T Cells after Hematopoietic Stem Cell Transplantation Prompts Thymic Renewal in Adult Patients by an IL-7 Dependent Mechanism
Luca Vago,1,2 Giacomo Oliveira,1 Maddalena Noviello,1 Corrado Soldati,3 Domenico Ghio,3 Immacolata Brigida,4 Alessandro Aiuti,4 Maria Tersa Lupo Stanghellini,2 Jacopo Peccatori,2 Attilio Bondanza,1 Alessandro Del Maschio,3 Claudio Bordignon,5,6 Fabio Ciceri,2 Chiara Bonini.1
1 Experimental Hematology Unit, San Raffaele Scientifi c Institute, Milan, Italy; 2 Hematology and BMT Unit, San Raffaele Scientifi c Institute, Milan, Italy; 3 Department of Radiology, San Raffaele Scientifi c Institute, Milan, Italy; 4 Pediatric Immunohematology and Bone Marrow Transplant Unit and San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), San Raffaele Scientifi c Institute, Milan, Italy; 5 ”Vita-Salute” San Raffaele University, San Raffaele Scientifi c Institute, Milan, Italy; 6 Molmed SpA, Milan, Italy.
In haploidentical Hematopoietic Stem Cell Transplantation (HSCT), the infusion of donor lymphocytes genetically modifi ed to express the Herpes Simplex Virus Thymidine kinase (HSV-Tk) suicide gene allows GvHD control, while rapidly providing an effective and polyclonal T cell repertoire against pathogens and underlying
malignancies (Ciceri and Bonini et al., Lancet Oncology, 2009) In the
TK007 phase I/II clinical trial 28 adult patients (median age: 50 years) with hematologic malignancies received purifi ed HSV-Tk-transduced cells after T cell-depleted HSCT, and 22/28 experienced a rapid and stable T cell immune reconstitution Even though their engraftment
is necessary to achieve these effects, HSV-Tk+ cells represent the minority of lymphocytes circulating in treated patients Therefore, we hypothesized an indirect role of HSV-Tk+ cells in prompting T cell development from graft progenitors by a thymus-dependent pathway
Trang 2Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
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RNA VIRUS VECTORS
T cell reconstitution of treated patients demonstrated recovery of
nạve HSV-Tkneg T cells The newly reconstituted CD4+ nạve T cells
were almost entirely comprised by CD31+ recent thymic emigrants
Accordingly, CT scans documented an increase in thymic volume
and single joint T cell Receptor Excision Circle counts rose following
HSV-Tk cell add-backs Comparison with a cohort of patients
subject to T-cell replete HSCT further suggested a unique direct role
of HSV-Tk+ cells in promoting thymopoiesis Interestingly, serum
levels of IL-7 markedly rose after Tk-cell add-backs, suggesting that
the genetically manipulated T cells may mediate the release of this
stromal cytokine, in turn supporting the generation and maturation
of T cells Notably, in the absence of HSV-Tk cell engraftment, no
increase in IL-7 serum levels was observed and patients did non
achieve the immune reconstitution The newly generated HSV-Tkneg
T cells granted persistent immune competence against infectious
agents, which was not compromised in those patients in whom the
suicide gene was activated to control GvHD These data show that
the infusion of suicide gene-modifi ed T cells induces IL-7 release,
boosts the function of the adult thymus and prompts the recovery of a
polyclonal, fully competent, T cell repertoire A phase III clinical trial
(TK008 study) to assess the effi cacy of HSV-Tk+ cells in the context
of haploidentical HSCT for leukemia started in Italy, and is currently
expanding to multiple centers throughout Europe and US
RNA Virus Vectors
Integration on Splicing of Cellular Genes at the
Genome-Wide Level
Stefania Merella,1 Jacopo Sgualdino,1 Daniela Cesana,1 Fabrizio
Benedicenti,1 Simone Leo,2 Gianluigi Zanetti,2 Luigi Naldini,1
Eugenio Montini.1
1 San Raffaele Telethon Institute for Gene Therapy, Milan, Italy;
2 Center for Advanced Studies, Research, and Development in
Sardinia, Pula, Italy.
Oncogenesis induced by insertional mutagenesis with gene therapy
vectors occurs mainly by activation of proto-oncogenes found
at or nearby the insertion site This activation often occurs by an
enhancer-mediated mechanism or by a process of splicing capture
which generates chimeric transcripts comprising portions of vector
and cellular mRNAs Although the activation of oncogenes may be
reduced by the use of self-inactivating (SIN) design and moderate
cellular promoters, how to reduce genotoxic splicing capture events
and aberrant transcript formation triggered by vector integration is
still unclear We developed a modifi ed Linear Amplifi cation-Mediated
(LAM) PCR technique, named cDNA LAM PCR (cLAM-PCR),
aimed at retrieving, from the whole transcriptome of LV-transduced
cells aberrantly spliced mRNAs that contain lentiviral vector (LV)
sequences fused with cellular transcripts in a high-throughput fashion
The sequences of cLAM-PCR products were obtained by 454
pyrosequencing and analyzed by a purposely build high-throughput
computational pipeline Our pipeline is based on a map-reduce
parallelization model, running in a private computer cluster and use
a dynamic analysis process composed by different steps implemented
as map-reduce applications Thus, chimeric LV-genome sequences
are recognized, the nucleotide position of the fused sequence is
identifi ed (the splice site), and the remaining portion mapped on
the appropriate genome assembly by BLAST Results obtained
with different LV constructs show that integrated LVs can perturb
the processing of cellular transcripts by interacting with the cellular
splicing machinery and fusing with its own splice sites to cellular
splice sites both upstream and downstream the integration site So
far, 70 different fusion transcripts could be identifi ed in total, 84%
of which were fused to known splice sites of gene exons, 6% were
fused to uncharacterized cryptic splice sites located in introns and the
remaining 10% were fused to genomic sequences not corresponding to any annotated gene We identifi ed several established and previously unknown splice sites within the LV backbone that participate in the aberrant splicing process with variable effi ciency Quantitative PCR on different portions of the LV backbone allows measuring the relative contribution to the aberrant splicing process of each
LV splice site identifi ed The amount of transcription occurring in regions outside the expression cassette reaches up to the 3% of the entire transgene expression The cLAM-PCR technique, coupled to high-throughput sequencing and the computational power of our specialized data analysis pipeline allows gaining insights into the biology of vector-mediated splicing alteration Since this process could induce neoplastic transformation by the generation of aberrant oncogenic protein, its in-depth characterization is instrumental in the development of next-generation LV with a higher safety profi le
607 Read-Through/Splicing-Capture Mechanism Is the Major Determinant of Enhancer Genotoxicity of Vectors with Active LTRs
Daniela Cesana,1 Marco Ranzani,1 Cynthia Bartholomae,2 Monica Volpin,1 Stefania Merella,1 Fabrizio Benedicenti,1 Lucia Sergi Sergi,1 Christof von Kalle,2 Manfred Schmidt,2 Luigi Naldini,1
Eugenio Montini.1
1 San Raffaele Telethon Institute for Gene Therapy, Milan, Italy;
2 National Center for Tumor Diseases, Heidelberg, Germany.
We recently developed and validated a new in vivo genotoxicity assay based on systemic vector injection into newborn tumor-prone Cdkn2a-/- mice to address the genotoxic potential of different VSV-G pseudotyped lentiviral vectors (LV) Treatment with an LV with self-inactivating (SIN) Long Terminal Repeats (LTRs) and harboring the strong Spleen Focus Forming Virus (SF) enhancer/promoter sequences in internal position driving GFP expression (SIN.LV.SF GFP), caused a signifi cant acceleration (p<0.006) in the time of tumor onset (N=17, 186 days median survival) with respect to mock-controls (N=80, 233 d) These data confi rm the higher sensitivity to vector genotoxicity of this new in vivo platform since this vector was neutral
in the previously validated Cdkn2a-/- transduction/transplantation mouse model Injection of an LV containing the same SF sequences within the LTR (LV.SF.LTR) caused a signifi cantly earlier tumor onset even with respect to the SIN.LV.SF.GFP-treated mice (N=11,
138 d, p<0.0001) These results provide a further confi rmation that the position of the enhancer/promoter sequences has a strong impact
on the genotoxic potential of integrative vectors To understand the reasons at the base of the different genotoxic potential between vectors with the enhancer in internal position or within the LTR, we deleted from the SIN.LV.SF.GFP the Open Reading Frame (ORF) downstream the SF promoter (LV.SF) and tested the effect of this modifi cation in the new in vivo platform Strikingly, LV.SF induced
a dramatic acceleration in the time of tumor onset (N=9, 112 d;
Vs Mock p<0.0001) similar to the LV.SF.LTR This data indicate that the presence of the ORF downstream the enhancer/promoter sequences and not the position or the number of enhancers per se has
a major role in increasing the safety profi le of the SIN design To get more insights into the mechanism responsible for this difference in genotoxicity, we retrieved the vector integration sites from tumors Analysis of >5000 LV integrations showed the presence of several Common Insertion Sites in all the different vector treatment groups, such as the well known cancer genes Braf, Sfi 1, Mef2c and Map3k8 Thus, all genotoxic vectors tested promoted tumor onset acceleration
by insertional mutagenesis Furthermore, the LV.SF.LTR and the LV.SF produced with high frequency chimeric LV/Braf transcripts
by a read-through/splicing-capture mechanism using available splice donor signals present in the vector or in the surrounding cellular genome On the other hand, no LV/Braf transcripts could be detected
in SIN.LV.SF.GFP induced-tumor indicating that the presence of an