203 Extended Duration of INFRADURE Biopump Secreting IFNα in Mice Shows Potential for Prolonged Therapeutic Effect in Patients Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The Amer[.]
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carrying OTC gene mutations had higher transaminase elevations
and loss of transgene in the presence of systemic infl ammation
However, in the absence TLR9 ligands, OTC gene mutations had
no signifi cant impact on the loss of vector genome copies Thus,
OTC gene mutations could predispose a gene therapy trial subject
to activation of a destructive cytotoxic T lymphocyte response in the
presence of systemic infl ammation
201 Factors Involved in the Induction of
Ag-Specifi c Immune Tolerance By
Hepatocytes-Directed LV.ET.mir142T Gene Transfer
Andrea Annoni,1 Mahzad Akbarpour,1,2 Luigi Naldini,1,2 Maria
Grazia Roncarolo.1,2
1 San Raffaele Telethon Institute for Gene Therapy (HSR-Tiget),
Milan, Italy; 2 Vita-Salute San Raffaele University, Milan, Italy.
We developed a lentiviral vector (LV) platform for
hepatocyte-directed gene expression, which combines two layers of transgene
regulation: hepatocyte-specifi c promoter (ET), and miR-142-targets
(142T) LV.ET.142T platform induces active tolerance towards the
encoded-antigen (Ag) via de novo generation of Ag-specifi c FoxP3+
peripheral T regulatory cells (pTregs), and partial deletion of
Ag-specifi c CD8+ effector T cells Gene transfer mediated by LV.ET.142T
led to stable gene replacement and suppression of FIX neutralizing
response in Hemophilia B mice In addition, inhibition of type-1
diabetes development in NOD mice was observed after gene transfer
with LV.ET.142T encoding for an insulin peptide
We investigated the cellular and molecular mediators underlying
the induction of pTregs and the deletion effector T cells, which led to
Ag-specifi c tolerance in mice treated with the LV.ET.142T platform
Differentiation of Ag-specifi c pTreg requires Ag-presentation in the
presence of transforming growth factor-beta (TGF-beta) In vitro
stimulation of CD4+ T cells revealed that all the studied hepatic
non-parenchymal cell subsets were capable to perform Ag-presentation
TGF-beta was up-regulated in the serum of the LV-tolerized
mice compared to LV-immunized or untreated controls, and gene
expression analyses indicated hepatocytes as the only hepatic cell
subset up-regulating tgf-beta transcription 7 days after LV.ET.142T
in vivo gene transfer Therefore, hepatocytes may represent the
source of TGF-beta in the liver microenvironment at the time of Ag
presentation, promoting pTreg differentiation
In addition to tgf-beta, hepatocytes also signifi cantly up-regulated
the expression of pdl-1 (Programmed cell death ligand 1), cIIta (class
II trans-activator), and h2ab1 (class II major histocompatibility
complex, MHC-II) To identify promoting factors for these genes,
primary hepatocytes were kept in culture for 48 hours with cytokine
related to immune response to LV, such as IL6, alpha and
IFN-gamma, alone or in combination with LV Results revealed that
IFN-gamma per se can up-regulate transcription of those genes
IFN-gamma-induced expression of PDL-1 was confi rmed at protein
level Therefore, PDL-1-PD1 co-stimulatory pathway was blocked
in Balb.c mice by anti-PDL-1 mAb after LV.ET.GFP.142T Three
weeks post LV injection an increase in GFP-specifi c CD8+ T effector
cells and a signifi cant reduction in GFP expressing hepatocytes was
observed These results indicate that PDL-1-PD1 co-stimulatory
pathway plays a role in promoting the contraction of transgene specifi c
CD8+ T cells response
Overall these data indicate that the tolerogenic program induced
by hepatocyte-targeted gene transfer requires partial activation of
the effector CD8+ T cells These effector cells incompletely primed
by hepatocytes release IFN-gamma, which up-regulates PDL-1,
dampening the effector response
202 Nucleic Acid-Binding Polymers Regulate Infl ammatory Disorders Without Causing Overall Immune Suppression
Eda Holl,1 Kara Shumansky,1 Bruce Sullenger.1
1 Department of Surgery, Duke University, Durham, NC.
Toll-like receptor (TLR) family members, TLR 3, 7 and 9 are key components in initiation and progression of autoimmune disorders and along with their downstream signaling pathways, they have become one of the main drug targets for disease amelioration During autoimmune disease progression, such as in the case of systemic lupus erythematosus (SLE), these receptors recognize self nucleic acid complexes and contribute to infl ammatory cytokine production and subsequent enhancement of serum autoantibody levels We have recently discovered a new class of nucleic-acid scavenging agents that can neutralize the proinfl ammatory effects of nucleic acids on a variety
of immune cells implicated in autoimmune disease development We have shown that these nucleic acid-scavenging polymers can inhibit TLR activation and subsequent cytokine production by both dendritic cells (DCs) and B cells of wild type and lupus prone mice Moreover, several of these agents can block nucleic acid-driven plasma cell differentiation and antibody production In vivo, we have shown that nucleic-acid binding polymers can prevent skin lesions following mechanical injury in lupus prone animal models Stimulation of immune cells by encapsulated viral particles is unaffected in the presence of polymers These fi ndings are further supported by an in vivo model of murine infl uenza Infected animals treated with polymer undergo a normal disease progression, and their immune response
to said infection remains fully intact Thus showing that the effects observed in the initiation and maintenance of the immune response
in the presence of polymers is specifi c to nucleic acid stimulation and not overall immune suppression These fi ndings provide a new avenue in drug development as these agents can potentially be utilized
to block overt autoimmune disorders without compromising normal immune responses
203 Extended Duration of INFRADURE Biopump Secreting IFNα in Mice Shows Potential for Prolonged Therapeutic Effect in Patients
Reem Miari,1 Tamar Shatil,1 Yael Grimpel,1 Osnat Tal,1 Atar Liron,1 Mati Metzuyanim,1 Simcha Krispel,1 Philip Ng,2 Nir Shapir.1
1 Medgenics Medical Israel Ltd, Misgav, Israel; 2 Dept of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX.
Autologous Biopumps are processed from dermis needle biopsies, with typical dimensions of 30 mm by 2 mm diameter These Biopumps harvested from the patient’s skin under local anesthesia, are transduced ex-vivo with Helper Dependent Adenoviral vector (HDAd) containing target protein expression cassette This process enables the Biopumps to continuously secrete the desired patient’s defi cient protein
Previously we have reported months of sustained elevated hemoglobin levels from a single subcutaneous implantation of EPODURE Biopumps secreting erythropoietin (EPO) in anemic patients with renal failure in the absence of exogenous EPO injections However, protein expression from implanted Biopumps could be limited post-implantation by various factors, including local infl ammation, reducing metabolic function, loss of gene copies,
or expression cassette instability To minimize these factors and prolonging the expression duration, two improvements were made: i) anti-infl ammatory agent, Depo-Medrol (DM), was applied along with the Biopump implantation and ii) new HDAd gene expression cassette was designed to increase stability of expression
In this study we report the in-vivo and in-vitro performance of INFRADURE Biopumps containing the HDAd-CAG-opt-hIFNα (control) or the CpG free stabilized expression cassette:
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EF1α-opt-hIFNα Biopumps transduced with viral vectors containing
each of the expression cassettes were implanted subcutaneously into
SCID mice, with or without administration of DM at the implantation
site Without DM, human Interferon-α (hIFNα) serum levels of mice
implanted with Biopumps transduced with the stabilized vector
remained to secrete 6.3% of initial peak at about 100 days post
implantation, whereas only about 1% of the initial peak levels was
observed in the control vector group, measured less than two months
post implantation When DM was applied, hIFNα serum levels were
improved to 19% of initial peak at about 100 days post implantation
with the stabilized vector, whereas no major improvement in duration
of secretion was observed with the control vector
Since our clinical studies to date have used the control vector
without DM, these results suggest that Biopumps transduced with the
combination of stabilized expression cassette and DM may secrete
sustained therapeutic levels of target proteins and could prolong and
enhance the therapeutic effect in patients
204 Research on Molecular Chimeric Cells
Infusion To Reduce Spleen T Lymphocyte
Response To Allogeneic T Cells Stimulation
Weidong Li,1 Weihua Fu,1 Li Lu,1 Ningbo Liu,1 Jiangong Cui,1
Liwei Zhu.1
1 General Surgery Department, Tianjin Medical University General
Hospital, Tianjin, China.
Objective: To construct the mouse bone marrow hematopoietic
stem cells (BHSCs) and Pre-T cells with chimeric MHC-I gene, and
to explore the mechanism of them reducing spleen T lymphocyte
response to allogeneic mouse T cells Methods: The mouse (BALB/c)
BHSCs and Pre-T cells were collected The identifi ed BHSCs and
Pre-T cells were transfected by the constructed eukaryotic expression
vector of C57BL/6 mouse MHC-I (pIRES-H-2Db and pIRES-H-2Kb)
Then the molecular chimeric cells were transfused back BALB/c
mouse After 7 days, the ability of molecular chimeric cells inducing
spleen T lymphocyte response to allogeneic T cells was observed
through mixed lymphocyte culture (MLC) Results: The BALB/c
mouse BHSCs and Pre-T cells were successfully separated and
cultured Flow cytometry analysis indicated that H-2Db and H-2Kb
protein expression were (25.20±0.88)% and (34.20±0.46)% in BHSCs
group, and in Pre-T cells group, H-2Db and H-2Kb protein expression
were (14.90±0.56)% and (14.20±0.63)% respectively
The result of MLC demonstrated that the stimulation index (SI) of
T lymphocyte were signifi cantly decreased in molecular chimeric cells group(P <0.01) The SI of T lymphocyte was even lower in
Pre-T cells group than in BHSCs group(P <0.01) Conclusion: The
molecular chimeric BHSCs and Pre-T cells infusion could reduce spleen T lymphocyte response to allogeneic T cells The molecular
chimeric Pre-T cells show better therapeutic effects [Key words]
BHSC; Pre-T cell; MHC-Igene; Molecular chimerism
205 Chemical Modifi cation of Messenger RNA Alters Their Activity in Cultured Cells
Anton P McCaffrey,1 Julie R Escamilla-Powers,1 Anthony C Lauer,2 Laura K Juckem.2
1 TriLink BioTechnologies, San Diego, CA; 2 Mirus Bio, Madison, WI.
Recently, there has been significant interest in the use of messenger RNA (mRNA) based expression systems for gene therapy applications, as well as for the generation and manipulation of stem cells Several groups have shown that mRNA is an attractive vehicle for therapeutic gene expression in mammals (Kormann et al Nat Biotechnol (2011) 29, 154; Kariko et al Molecular Therapy (2012)
20, 948) Additionally, Warren et al demonstrated highly effi cient induced pluripotent stem cell (iPS cells) generation by transfection
of mRNAs encoding reprogramming factors (Warren et al Cell Stem Cell (2010) 7, 618) mRNA poses no risk of insertional mutagenesis and subsequent oncogenesis For this reason, the authors suggested that iPS cells generated in this manner should be safer than iPS cells derived by plasmid transfection or viral transduction In addition, transient expression from mRNA is desirable for applications such
as genome editing using zinc-fi nger nucleases, TALENs and Cas9/ CRISPR
A key insight for the development of mRNA expression systems was the recognition that mRNA induces innate immune responses
in transfected cells Kariko et al showed that substitution of uridine and cytidine residues with pseudouridine and 5-methylcytidine dramatically reduced innate immune recognition of mRNA (Kariko
et al Molecular Therapy (2008) 16, 1833) They also showed that pseudouridine modifi ed RNA was translated more effi ciently and had increased nuclease resistance
These studies highlight the importance of the development of stable, non immunogenic mRNA Previous studies have explored different patterns of chemical modifi cation in mRNA but it is diffi cult to draw conclusions since activity between studies cannot be compared We have compared selected combinations of chemical modifi cations in cultured cells We found the effect of specifi c chemical modifi cations