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San Raffaele Hospital and Scientific Institute
Via Olgettina 60
Milan, Italy
www.hsr.it
The Office of Biotechnology Transfer, established in 1992, is acting as the interface between the
San Raffaele Hospital and Scientific Institute and the business community in the life science
sector (pharma groups, biotech companies, food and cosmetic industries) as well as merchant
banks and venture capitalists.
The mission of the Office of Biotechnology Transfer is to create value from know-how, intellectual
property, human resources and research facilities available within the San Raffaele Biomedical
Science Park, which includes the San Raffaele Hospital (the most renowned Italian hospital, with
1400 beds, around 276 clinical trials per year), the San Raffaele Scientific Institute (DIBIT), one
of the biggest in Europe with around 1640 individuals distributed throughout the 6 research
Divisions, the 2 Research Centres and the 8 institutional Facilities, 1142 publications and an
impact factor of 6,451 in 2013, and the Vita-Salute San Raffaele University, offering a medical
school with residency programs and PhD courses in Molecular Medicine and Cellular and
Molecular Biology and faculties of Psychology and Philosophy.
The Office of Biotechnology Transfer is located within the San Raffaele Hospital and Scientific
Institute, in the eastern side of Milan, well connected to downtown via an internal underground
metro station, to the motorway that surrounds the city, close to the Milan Linate International
airport, with an internal heliport.
The Office of Biotechnology Transfer’s mission is to create value from the innovation generated
by scientific activities of the San Raffaele Hospital and Scientific Institute and promoting
partnerships with pharma and biotech companies.
Main activities of the Office of Biotechnology Transfer are:
vIntellectual property management
vPromotion of research results towards the life science w/w business community
vNegotiation and management of sponsored research projects
vLicensing of intellectual property rights
Important research contracts and licensing agreements have been recently signed with: GSK,
Merck-Serono, Novartis, Teva, Sangamo Biosciences, Biogen Idec, Dompè, ST
Microelectronics, MolMed.
The Office of Biotechnology Transfer has filed 141 patent families (patented technologies), on
behalf of the San Raffaele Hospital and Scientific Institute of which 48 (corresponding to around
220 patents and patent applications, in portfolio) are still alive, at 2014.
The technology transfer activities have produced the following results, at 2014:
v381
v70
v367
v320
v140
sponsored research and service contracts
license/option/evaluation agreements and amendments
confidentiality agreements with industrial partners
material transfer agreements with industrial partners
industrial clients worldwide (companies that signed research contracts and/or
license agreements)
For further information:
Office of Biotechnology Transfer
San Raffaele Hospital and Scientific Institute
Via Olgettina, 60 - 20132 Milan, Italy
Phone + 39 02 2643 4882
Fax + 39 02 2643 5264
www.hsr.it
2
Patent Index
CANCER
“Human Tie2-expressing proangiogenic monocytes (TEMs)”
(cancer angiogenesis, drug screening)
Page 5
“Tumor targeted conjugable peptides”
(tumor targeting peptides, therapeutic, diagnostic uses)
Page 7
“Biomarkers of multiple myeloma development and progression”
(metabolic profiling, multiple myeloma, diagnostic/prognostic/companion tools)
Page 9
“Targeting leukemia by CD1c-restricted T cells specific
for a novel lipid antigen”
(leukemia immunotherapy, tumor-associated lipid antigens)
Page 11
“Novel targets in multiple myeloma and other disorders”
Page 13
(DNA damage, synthetic-lethal therapy, multiple myeloma and other hematological disorders)
IMMUNE DISEASES
“Clinical use of Rapamycin-expanded regulatory-T cells
for the treatment of T-cell mediated diseases”
(cell therapy of immune disorders, GvHD, autoimmune diseases, type I diabetes)
Page 15
“Tr1-dendritic cells and uses thereof”
(cell therapy of immune disorders, GvHD, autoimmune diseases, type I diabetes)
Page 17
METABOLIC DISEASES
“IGFBP3 and uses thereof”
(gastrointestinal disorders, type 1 diabetes, therapeutic/diagnostic uses)
Page 19
STEM CELLS AND REGENERATIVE MEDICINE
“Periangioblasts, adult skeletal muscle stem cells
for the treatment of muscular dystrophies”
(stem cell therapy of muscular dystrophies, skeletal muscle)
“Use of neural stem cells to induce neuroprotection
in inflammatory CNS disorders”
(stem cell therapy of CNS inflammatory disorders, multiple sclerosis,
neurodegeneration, inflammation)
“A non-oxidable HMGB1 mutant for wound healing”
(tissue regeneration, wound healing)
Page 21
Page 23
Page 25
3
GENE THERAPY
“MicroRNA-regulated Viral Vectors”
(gene therapy, research tool)
Page 27
“New promoters and new lentiviral vectors:
efficient and coordinated expression of multiple genes”
(gene therapy, research tool)
Page 29
“A method for the ex vivo production of fully functional
gene-modified human T lymphocytes”
(gene therapy, cancer)
Page 31
“New gene therapy approach to induce antigen-specific
immunological tolerance”
(gene therapy, gene delivery)
Page 33
NEUROSCIENCE
“Myeloid Microvescicles are a marker and therapeutic target
for neuroinflammation”
(multiple sclerosis, Alzheimer, biomarkers, therapeutic target)
“PTGDS pathway activators and uses thereof”
(peripheral demyelinating diseases, therapeutic/diagnostic uses)
Page 35
Page 37
MEDICAL DEVICE
“Composite Scaffold for Tissue Repair”
(orthopedics, osteochondral, rigeneration)
Page 39
TECHNOLOGIES
“Optical platform for ion channel drug screening”
(optical method, HTS, ion channel)
Page 41
FOOD
“Low carbs arginine bar”
(Arginine, obesity, diabetes, weight loss)
Page 43
4
“Human Tie2-expressing proangiogenic monocytes (TEMs)”
Human CD14+ monocytes can be divided into two main subsets according to the expression of CD16, a Fc
gamma receptor III. CD14highCD16– cells are the most abundant monocytes in peripheral blood and are
thought to represent classical monocytes that mediate inflammatory responses (‘inflammatory’ monocytes),
whereas CD14lowCD16+ cells are a less characterised subset, which are thought to represent the
precursors of tissue-resident macrophages and are referred to as ‘resident’ monocytes. Interestingly, the
inventors found that a subset of CD14lowCD16+ monocytes expressed the TIE2 angiopoietin receptor.
These Tie2-expressing monocytes (TEMs), but not the other monocytes, markedly promoted angiogenesis
in xenotransplanted human tumours. In human cancer patients, TEMs were observed in the blood and
were specifically recruited to the tumors, where they represented the main monocyte population distinct
from tumour-associated macrophages (TAMs). In vitro, human TEMs migrated towards Angiopoietin-2
(ANG2), a TIE2 ligand and proangiogenic factor released by activated endothelial cells and angiogenic
vessels (Venneri et al., Blood. 2007; De Palma M et al., Trends Immunol. 2007). In mouse tumor models,
TEMs promote angiogenesis by associating with sprouting blood vessels (De Palma M et al., Cancer Cell.
2005. De Palma M et al., Cancer Cell. 2008; Pucci F et al., Blood. 2009). The inventors found that ANG2
blockade regresses the tumor vasculature and inhibits progression of late-stage, metastatic tumor models.
Although ANG2 blockade did not inhibit TEM recruitment to the tumors, it impeded TEM’s upregulation of
TIE2, their association with blood vessels and ability to promote tumor angiogenesis. Furthermore,
conditional Tie2 gene knockdown in TEMs was sufficient to decrease tumor angiogenesis (Mazzieri et al.,
Cancer Cell. 2011; De Palma M, Naldini L. Clin Cancer Res. 2011). Thus, TEMs represent important
regulators of tumor angiogenesis.
Human TEMs may provide a novel, biologically relevant marker of angiogenesis and represent a
previously unrecognized target of cancer therapy.
Patent granted in US (US7833789) covers human proangiogenic cells, and pharmaceutical
compositions thereof.
TEMs are a subset of ‘resident’ monocytes that are found in human tumors
25% 6%
Empty
CD16
4%
70%
TIE2
21%
CD14
IgG
11%
Figure left side. Expression of CD14 and CD16
identifies two distinct monocyte subsets. The
gated cell populations (stained in different colors)
were analysed for expression of TIE2 (upper right
dot plots) versus isotype control (lower right dot
plots). Note that the CD14lowCD16+ fraction
(resident monocytes; red dots) is highly enriched
in TIE2+ cells, whereas the CD14+CD16– fraction
(inflammatory monocytes; violet dots) contains
few TIE2+ cells.
Figure above. TEM recruitment to tumors. A, a human glioblastoma orthotopically injected in the mouse
brain shows several TEMs (labeled by green fluorescent protein, green) associated with the tumor blood
vessels (red, stained by an anti-CD31 monoclonal antibody). B, a human gastric adenocarcinoma stained
with an anti-Tie2 monoclonal antibody. Note the Tie2+ blood vessels and the presence of scattered Tie2+
mononuclear cells (TEMs, arrows) in the stroma. Section was counterstained by H&E. C, a human colon
carcinoma stained with a fluorescent anti-Tie2 monoclonal antibody (red) shows Tie2+ blood vessels and
scattered Tie2+CD14+ TEMs (arrows). Green, CD14+ monocytes were stained with a FITC-conjugated
anti-CD14 monoclonal antibody. Double-positive cells (i.e., TEMs) appear yellow. Bar, 50 Am
5
Stage of Development
They show that human TEMs are distinct from classical proinflammatory cells, migrate towards
Angiopoietin-2, are preferentially recruited to tumors, and display marked proangiogenic activity.
Interfering with Tie2 expression in TEMs, or neutralizing the TIE2 ligand, ANG2, limits the formation of
intratumoral vascular networks. Mazzieri et al. (2011) recently showed that ANG2 blockade:
(i) inhibits angiogenesis and induces vascular regression in multiple tumor models, including tumors that
are prone to develop resistance to anti- VEGF/VEGFR therapy; (ii) inhibits tumor growth in multiple tumor
models, including late-stage spontaneous tumors; (iii) limits the metastatic dissemination of primary
tumors and the outgrowth of established metastasis; and (iv) impedes, in tumor-infiltrating TEMs, the
transcriptional upregulation of Tie2, which is required for their association with tumor blood vessels and
proangiogenic activity.
Competitive Advantages
Among other circulating Tie2+ proangiogenic cells such as circulating endothelial cells (CECs) and
progenitors (EPCs), TEMs should represent ideal candidates to monitor/target angiogenesis, for the
following reasons:
• TEMs are more abundant in the peripheral blood than the elusive CECs
• TEMs can be easily distinguished from other hematopoietic cells subsets by the combination of at least
three surface markers.
• TEMs circulate in the peripheral blood, thus they could be assayed by a simple procedure. TEMs might
serve as a quantitative pharmacodynamic marker to monitor the angiogenic phenotype in a living
organism or a patient and the effectiveness of antiangiogenic therapies.
• TEMs have superior proangiogenic activity among a number of hematopoietic cell populations tested,
including classical inflammatory monocytes and CECs/EPCs. Furthermore, TEMs are committed to a
proangiogenic function already when they circulate in the peripheral blood, appearing to be a distinct
lineage of previously unknown cells with dedicated (proangiogenic) function. Thus, TEMs may be targets
of novel cancer therapies, once molecular features are identified that selectively distinguish the activity of
these cells.
• Strategies to deplete TEMs or reprogram them toward an angiostatic/proinflammatory phenotype may
enhance the therapeutic activity of various anticancer therapies and counteract tumor resistance to
chemotherapy (Squadrito, M.L. & De Palma, M. Mol Aspects Med. 2011).
Potential Applications
The identification of human Tie2+ monocytes may open a number of avenues in the development of novel
anticancer therapies. Tie2+ monocytes may represent targets of novel antiangiogenic therapies and
biological readouts in the circulation to monitor pathological angiogenesis.
Furthermore, TEMs can be used as gene delivery vehicles in the setting of an autologous bone marrow
transplant or adoptive transfer (De Palma M et al., Cancer Cell. 2008).
We seek a commercial partner with a strong pipeline in angiogenesis/cancer therapeutics to
further explore TEM’s depletion or reprogramming to angiostatic/proinflammatory phenotype to
enhance the therapeutic activity of anticancer therapies, and counteract tumor resistence to
chemotherapy.
For further information on this project please contact:
Business Contact
Paola Pozzi
Head, Office of Biotechnology Transfer
San Raffaele Hospital and Scientific
Institute
Tel: +39 02 2643 4987
Fax: + 39 02 2643 5264
Email: [email protected]
Scientific Contact
Prof. Luigi Naldini
Director San Raffaele Telethon Institute for Gene
Therapy
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 4681
Fax: + 39 02 2643 4621
Email: [email protected]
6
“Tumor targeted conjugable peptides”
Background
Herein, the inventors propose to increase the tumor homing properties of albumin-based drugs and
nanoparticles by an "active" targeting mechanisms, by coupling albumin with ligands selective for
receptors overexpressed in the tumor vasculature such as αvβ3 heterodimer.
Because of its biocompatibility, its long circulating half-life and its tendency to accumulate in tumors
(owing to increased permeability and defective lymphatic drainage in neoplastic tissues) albumin is
emerging as a versatile drug carrier in a number of applications in cancer therapy.
Notably, albumin-paclitaxel nanoparticles (Abraxane), have been approved for the treatment of metastatic
breast cancer, (www.abraxane.com) highlighting the importance of this protein as a versatile material for
the successful development of new anticancer nanomedicines.
Description of Invention
The present invention is a new head-to-tail-cyclized hexapeptide containing the isoAsp-Gly-Arg
(isoDGR) motif that, after chemical conjugation to human serum albumin (HSA), recognizes αvβ3
with very good selectivity, binds to tumor vessels, inhibits tumor growth and works as an efficient
ligand for the delivery of nanomedicines to tumor vasculature.
IsoDGR is a tripeptide sequence that can arise in fibronectin as a
consequence of spontaneous asparagine deamidation at Asn-GlyArg (NGR) sites and that works as a biological switch for the
regulation of cell adhesion. IsoDGR is a mimetic of Arg-Gly-Asp
(RGD), an important integrin recognition motif present in various
proteins involved in the regulation of cell adhesion. The inventors
and other investigators have shown that isoDGR can recognize RGD
dependent integrins (such as αvβ3, αvβ5, αvβ6, αvβ8 and α5β1) with
different affinity and selectivity, depending on isoDGR conformation
and molecular scaffold. To fulfill these aims the inventors have
designed a series of head-to-tail-cyclized isoDGR penta and hexapeptides containing a free thiol group and analyzed their integrin
binding properties before and after conjugation to proteins and
nanoparticles. Peptide-albumin conjugates were positively tested for
(i) integrin binding properties, (ii) capability to recognize the Figure 1. Gold nanoparticles (Au)
endothelial lining of tumor vessels and (iii) anti-cancer activity in loaded with isoDGR-albumin (1-HSA)
mouse fibrosarcoma and lymphoma models (Curnis, Corti et al. and TNF.
IsoDGR-tagged albumin: a new αvβ3 selective carrier for nanodrug
delivery to tumors. Small 2012).
Overall, the inventors have identified a cyclic hexapeptide (called isoDGR#1) that, after coupling to
human serum albumin (HSA), has a very good selectivity for αvβ3, binds to tumor vessels (Figure 2) and
inhibits tumor growth. Furthermore, in vivo studies in mice bearing WEHI fibrosarcomas showed that
coupling the isoDGR#1-HSA conjugate (called 1-HSA) to TNF-bearing gold nanoparticles (25 nm)
(Figure 1), a known tumor vessel damaging agent, enhanced the anti-tumor activity of this nanomedicine
more efficiently than coupling with HSA. Notably, doses of this nanomedicine equivalent to 5 pg of
bioactive TNF was sufficient to induce significantly delay of tumor growth whereas “non-targeted” TNF
was inactive (Figure 3).
Because of its good selectivity for tumor vessels and its inherent anticancer activity the 1-HSA conjugate
might be exploited as a novel and versatile material for the preparation of a wide range of tumor
vasculature-selective drugs and nanoparticles for cancer therapy and diagnosis.
The international patent application was published as WO2013140317. Patent applications
pending in Europe and US.
7
Figure 2.
1-HSA/Qdot,
but
not
HSA/Qdot, binds to endothelial lining of
tumor vessels on murine RMA lymphoma
tissue sections. Frozen sections were
incubated with 1-HSA, or control HSA,
chemically
coupled to fluorescent
quantum dot nanoparticles (1-HSA/Qdot
and ∗HSA/Qdot), and immunostained
with anti-CD31 antibody (a marker of
endothelial cells). Red, Qdots; blue,
DAPI; green, CD31.
Figure 3. Coupling 1-HSA to TNF-loaded gold nanoparticles could enhance their anti-tumor activity. Significantly
lower effects were observed with an equivalent dose of gold nanoparticle bearing TNF alone, or even with 10-fold
higher doses. Equivalent doses of free TNF were completely inactive.
Competitive Advantages
Higher selectivity. Considering that αvβ8 is expressed in yolk sac, placenta, brain perivascular astrocytes, Schwann
cells, renal glomerular mesangial cells and pulmonary epithelial cells and that αvβ6 is expressed in epithelia, the
higher selectivity of 1-HSA for integrins expressed in tumor vessels might represent an important advantage.
Interestingly, both linker and protein scaffold markedly contribute to the selective recognition of αvβ3 by 1-HSA.
Enhancement of binding affinity and selectivity was observed also after coupling peptide isoDGR #1 to avidin.
No toxicity. 1-HSA did not cause loss of body weight or evident toxic reactions at any tested dose.
These results, overall, suggest that isoDGR-tagged albumin is a new vascular targeting agent that might be exploited
in place of albumin for the preparation of new nanotherapeutics and nanodiagnostics with improved tumor homing
ability.
Of note, even the peptide isoDGR#1 (uncoupled, with a free thiol group) can be exploited in principle as a ligand for
the functionalization of a number of therapeutic and diagnostic compounds and nanoparticles thereby improving their
tumor homing ability (Corti, Curnis, et al. Peptide-mediated targeting of cytokines to tumor vasculature: the NGRhTNF example. BioDrugs. 2013)
We seek a potential commercial partner focused on tumor targeted therapies for enhancing
efficacy of anti-tumor drugs.
For further information on this project please contact:
Business Contact
Paola Pozzi
Head, Office of Biotechnology Transfer
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 4987
Fax: + 39 02 2643 5264
Email: [email protected]
Scientific Contact
Dr. Angelo Corti
Tumor Biology and Vascular Targeting Unit
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 4802
Fax: + 39 02 2643 4786
Email: [email protected]
8
“Biomarkers of multiple myeloma development and progression”
Background
Multiple Myeloma (MM) is a neoplastic disorder of plasma cells (PC), which typically grow at multiple foci
in the bone marrow (BM), secrete monoclonal immunoglobulins (Ig), and induce end-organ damage
leading to hypercalcemia, renal failure, anemia and bone lesions. Treatable, but still incurable, MM
accounts for 2% of all cancer deaths.
MM originates from MGUS (monoclonal gammopathy of undetermined significance), an asymptomatic
expansion of a PC clone occurring in 3% adults over 50 years, with 1-2% yearly risk of progression to
myeloma. An intermediate condition, smoldering myeloma (SMM) is defined by the presence of >10%
PCs in the BM or >3g/dl serum monoclonal Ig (M-component) in the absence of symptoms.
In light of the recent development of more effective therapies, the possibility to treat SMM patients is
currently under investigation. However, the great variability in timing and lifetime risk of progression
requires new sustainable follow-up strategies for early identification of individuals progressing to active
disease. New powerful and non-invasive prognostic tools are thus needed.
In search for novel markers of MM development and progression, inventors exploited metabolomics,
the systems biology of small molecules, to achieve an unbiased, comprehensive assessment of the
complete set of small metabolites within the extracellular BM milieu and peripheral plasma.
Inventors’ work demonstrates that peripheral metabolites define the metabolic changes associated with
development and progression of MM, leading to the identification of new markers of myeloma
progression, of prognostic value and pathogenic significance.
Description of Invention and Potential Applications
To achieve an unbiased, comprehensive assessment of the extracellular milieu of myeloma, our scientists
performed a metabolomic analysis of patient-derived peripheral and BM plasma by ultra high performance
liquid and gas chromatography followed by mass spectrometry (UHPLC-GC/MS). By multivariate
analyses, metabolic profiling of both peripheral and BM plasma successfully discriminated active disease
from control conditions (health, MGUS or remission). Noteworthy, the peropheral metabolome (assessed
in as little as 200 µl) correlated with BM PC counts, reporting on tumor burden.
Significant changes in the peripheral metabolome were associated to renal dysfunction, independently
from disease load. Non-overlapping disease vs. control analyses consistently identified a number of
metabolic alterations that hallmarked active disease, including increased levels of a complement peptide,
of uncommon aminoacids, and a fall in an entire class of lipids. These unanticipated markers desribe new
pathways associated with myeomagenesis, currently being tested. An exemplar case, in vitro tests on cell
lines and patient-derived MM cells revealed a previously unsuspected direct trophic function of those
lipids on malignant PCs.
The international patent application, covering the results of the studies, was published as
WO2014068144 and it is available for licensing worldwide.
Patent applications pending in Europe and US
9
Peripheral Metabolic Score
Diagnostic Group
Competitive Advantages
Importantly, the invention is a valuable prognostic tool to:
i) identify individuals with precursor conditions (MGUS and SMM) at high risk of developing active MM;
ii) predict response to treatment to design personalized therapeutic regimens;
iii) predict timing of the inevitable relapse in patients that responded well to therapy.
Stage of Development
Upon informed consent subscription, as approved by the institutional review board, 167 blood samples
were obtained from MM or MGUS patients at Ospedale San Raffaele from 2009 to 2011.
Inventors assessed the metabolic correlates of MM development and progression in plasma samples
from patients newly diagnosed with MM (NEW, n=16), with relapsing or progressive disease (PRO,
n=20), in clinical remission (REM, n=13), with MGUS (n=30), 25 with SMM (SMM, n=17) and from agematched healthy volunteers (HV, n=29).
Different analytical methods and independent comparisons of disease vs. non disease groups converged
in identifying a panel of discriminants with statistical significance in univariate analysis among groups.
A dedicated assay may be developed for the targeted profiling of a very set of metabolites which
have been identified by our scientists.
We seek a potential commercial partner focused on development of a dedicated biochemical
assay as a predictive tool for assessing risk of development and progression of Multiple Myeloma,
monitoring response to treatment and/or therapeutic efficacy, and predicting relapses.
For further information on this project please contact:
Business Contact
Paola Pozzi
Head, Office of Biotechnology Transfer
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 4987
Fax: + 39 02 2643 5264
Email: [email protected]
Scientific Contact
Dr. Simone Cenci
Division of Genetics and Cell Biology
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 6783
Fax: + 39 02 2643 4767
Email: [email protected]
10
“Targeting leukemia by CD1c-restricted T cells specific for a
novel lipid antigen”
Background
Acute leukemia comprises a heterogeneous group of hematological disorders characterized by blood and
bone marrow accumulation of immature and abnormal cells derived from hematopoietic precursors.
Current therapy for acute leukemia is based on poly-chemotherapy and allogeneic Hematopoietic Stem
Cell Transplantation (HSCT). A major cause of treatment failure in HSCT is post-transplant re-growth of
residual leukemia blasts that survive the conditioning regimen. Donor-derived T cells transferred into
patients may induce a beneficial Graft Versus Leukemia (GVL) reaction capable of maintaining remission,
but grafted T cells are also capable of killing patient cells in non-hematopoietic tissues inducing
detrimental Graft Versus Host Disease (GVHD).
To overcome this problem and improve the efficacy of the HSCT, herein, the inventors propose a new
immunotherapy strategy taking advantage of an immune recognition of specific tumor-associate lipid
antigens in order to selectively target T cell responses against malignant hematopoietic cells.
Description of Invention
The present invention described the identification of a previously unknown class of self-lipid
antigens, named as methyl-lysophosphatidic acids (mLPAs), which are usually accumulated in
leukemia cells and function as potent agonists for CD1c-restricted human T cells.
The immune system contains T cells that recognize lipid antigens presented by the non-polymorphic, MHC
class I-related family of CD1 molecules. CD1-restricted T cells can respond to different foreign lipid
antigens derived from pathological bacteria and can also recognize endogenous self-lipid molecules. T
cells that recognize self-lipids presented by CD1c are relatively abundant among circulating T cells in
healthy individuals and might become activated by host antigen in autoimmune disease and cancer.
Importantly, lipid-specific T cells can control cancer cell growth and kill transformed hematopoietic cells,
but little is known about their self-lipid antigen specificity and potential anti-leukemic effects.
In this respect, in the present invention, the inventors have identified the methyl-lysophosphatidic acids
(mLPAs), a novel self-lipid antigens that stimulates CD1c auto-reactive T cells to destroy tumor cell lines
and primary leukemia cells (Lepore M. et al., A novel self-lipid antigen targets human T cells
against CD1c(+) leukemias. J Exp Med. 2014). The inventors reported that blasts, derived from pediatric
and adult patients affected by primary acute myeloid or B-cell acute leukemia, express CD1c molecules
and that mLPAs accumulate in leukemia cells, but are poorly present in normal hematopoietic cells.
mLPA-specific T cells efficiently kill in vitro CD1c+ primary acute leukemia blasts, poorly recognizing nontransformed CD1c-expressing cells, and, in addition, protect immune-deficient mice against CD1c+ human
leukemia cells. The efficacy of mLPA-specific T cell clones to restrain the progression of human leukemia
grafted into NOD/scid mice is shown in Figure 1.
The identification of immunogenic self-lipid antigens accumulated in leukemia cells and the observed
leukemia control by lipid-specific T cells in vitro and in vivo provide a new conceptual framework for
leukemia immunosurveillance and possible immunotherapy.
The international patent application was filed.
11
A
B
Figure 1. (A) CD1c+ MOLT-4 acute leukemia cells (106cells/mouse) were injected i.v. into 15 immunodeficient
NOD/scid mice. After 48h, two groups of 5 mice each received 107 cells/mouse of the mLPAspecific T cell clone
K34A27f or the M. tuberculosis-specific, CD1crestricted T cell clone DL15A31. Mice receiving mLPA-specific T cells
displayed significantly increased survival compared to mice that received CD1c-restricted T cell clone specific for a
bacterial lipid, or vehicle alone (**p<0.02 Mantel-Cox test). (B) Primary CD1c+ AML blasts were injected i.v.
(8x106cells/mouse) into 14 NOD/scid/common γ chain-/- (NSG) mice. Two days later, mLPA-specific K34A27f T cells
(1.5x107cells/mouse) were injected into half of the mice and leukemia progression was monitored weekly by flow
cytometry of PBMCs. Also in this case, the leukemia progression was significantly delayed by the transfer of mLPAspecific T cells (P < 0.001, non-parametric Student t-test).
Competitive Advantages
Harnessing CD1c self-reactive T cell responses is an attractive option for adoptive immunotherapy of
leukemia especially in the context of Hematopoietic Stem Cell Transplantation (HSCT) for the following
several reasons:
• The restricted CD1c expression on hematopoietic cells minimizes the risk of Graft Versus Host
Disease (GVHD);
• The lack of CD1 polymorphisms permits to use allogeneic CD1 self-reactive T cell to treat any
leukemia patient;
• Since more than 50% of AML cases expresses CD1c molecules and are recognized by mLPA-specific
T cells, the frequency of patients that could benefit from this adoptive T cell immunotherapy would be
relevant;
• Different from MHC-restricted protein antigens, lipid antigens are unlikely to undergo structural
changes under the immune-mediated selective pressure, reducing the risk for leukemia immune
escape.
We seek a potential commercial partner with a strong expertise in cancer adoptive immunotherapy
to further explore the clinical use of lipid-specific T cells for the treatment of leukemia.
For further information on this project please contact:
Business Contact
Paola Pozzi
Head, Office of Biotechnology Transfer
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 4987
Fax: + 39 02 2643 5264
Email: [email protected]
Scientific Contact
Drs. Giulia Casorati & Paolo Dellabona
Experimental Immunology Unit
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 4727
Fax: + 39 02 2643 4786
Email: [email protected]; [email protected]
12
“Novel targets in multiple myeloma and other disorders”
Background
Multiple Myeloma (MM) is the second most frequent hematological cancer after non-Hodgkin’s lymphoma
and is characterized by the accumulation of neoplastic plasma cells in the bone marrow. Despite recent
advances in therapies and improved patient outcomes, MM remains an incurable cancer, hence novel
therapies are urgently needed.
Herein, the inventors propose a new synthetic-lethal strategy to treat MM and other hematological
cancers, including lymphoma and leukemia, by selectively targeting of cancer cells presenting with
endogenous DNA damage and low YAP1 levels.
Description of Invention
The present invention elucidates a synthetic-lethal approach in which genetic inhibition of serinethreonine kinase 4 (STRK4) reactivates the Hippo mediator YAP1, which, by interacting with ABL1,
triggers apoptosis in hematologic malignancies with intrinsic DNA damage, independently from
the mutational status of p53.
DNA damage elicits genomic instability in cancer cells. While epithelial cancer cells presenting DNA
damage inactivate tumor suppressor p53 to prevent the ensuing apoptosis, in hematological cancers the
relevance of ongoing DNA damage and the mechanism undertaken by hematopoietic cells to survive
genomic instability are largely unknown. The inventors identified a p53-indipendent network in MM and
other hematopoietic disorders centered on the nuclear relocalization of the pro-apoptotic ABL1 kinase as a
result of widespread DNA damage (Cottini F. et al., Rescue of Hippo coactivator YAP1 triggers DNA
damage-induced apoptosis in hematological cancers. Nat Med. 2014). In response to DNA damage,
nuclear ABL1 triggers cell death through its interaction with the Hippo pathway coactivator YAP1, which in
turn stabilizes p73 and coactivates p73 proapoptotic target genes. Nonetheless MM, lymphoma and
leukemia cells are able to survive by genetically inactivating or by exploiting the low expression levels of
YAP1. Gain-of-function studies show that increased YAP1 levels in hematological cancer cells promote
apoptosis by increasing the stability of the tumor suppressor p73 and its downstream targets, suggesting
that re-expression of YAP1 might trigger nuclear ABL1-induced apoptosis. YAP1 is under the control of a
serine-threonine kinase, STK4. Importantly, the inventors demonstrated that functional or pharmacological
inhibition of STK4 restores YAP1 levels and induces a robust apoptosis in vitro and in vivo, thereby
harnessing the ongoing DNA damage present in MM and other hematological cancer cells as a potential
Achille’s heel. Therefore novel therapies targeting STK4 now represent a promising novel therapeutic
strategy to improve patient outcome in MM and other hematological disorders.
An exemplification of the proposed model for the ABL-YAP1-p73 axis and the effects of STK4 inhibition on
YAP1 levels in hematological cancers is reported in Figure 1.
The international patent application was published as WO2014068542. Patent applications pending
in Europe and US.
13
Figure 1: Proposed model for the ABL-YAP1-p73 axis and the effects of STK4 inhibition on YAP1 levels.
Competitive Advantages:
• Druggability of the proposal target;
• Targeting MM patients with mutational status of p53.
We seek a potential commercial partner with a strong pipeline in kinase inhibitors in order to
develop new therapeutic agents for the treatment of multiple myeloma and other hematological
cancers.
For further information on this project please contact:
Business Contact
Paola Pozzi
Head, Office of Biotechnology Transfer
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 4987
Fax: + 39 02 2643 5264
Email: [email protected]
Scientific Contact
Dr. Giovanni Tonon
Functional Genomics of Cancer Unit
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 5624
Fax: + 39 02 2643 5602
Email: [email protected]
14
“Clinical use of Rapamycin-expanded regulatory-T cells for
the treatment of T-cell mediated diseases”
Rapamycin is an immunosuppressive compound currently used to prevent acute graft rejection in
humans. It is known that rapamycin allows operational tolerance in murine models. However, a direct
effect of rapamycin on the T regulatory (Tr) cells, which play a key role in the induction and maintenance
of peripheral tolerance, has not been demonstrated so far.
The naturally occurring Tr cells (CD4+CD25+FoxP3+) contribute to tolerance induction after solid organ
transplantation and protect from graft versus host disease (GvHD) lethality in bone marrow
transplantation models. Moreover, it has been recently observed that patients with autoimmune diseases
such as type 1 diabetes, multiple sclerosis, and rheumatoid arthritis are deficient in CD4+CD25+ Tr cells.
Scientist at DIBIT have established a method that selectively expands the naturally occurring
CD4+CD25+FoxP3+ Tr cells in vitro. In vitro long-term exposure of murine CD4+ T cells to rapamycin
induces expansion of the naturally occurring CD4+CD25+FoxP3+ Tr cells, which retain their suppressive
functions. The rapamycin-expanded Tr cells suppress T cell proliferation in vitro and prevent allograft
rejection in vivo. (Battaglia M, Stabilini A, Roncarolo MG. Rapamycin selectively expands
CD4+CD25+FoxP3+ regulatory T cells. Blood. 2005). Furthermore, rapamycin allows the selective
expansion and survival of human CD4+CD25+FOXP3+ Tr cells from periheral blood of both healthy
subjects and patients with type 1 diabetes (Battaglia M et al J.Immunology 2006). Recent data
demonstrate that a “pre-GMP” protocol for the expansion of human FOXP3+ Tr cells with rapamycin is
feasible and that rapamycin-expanded human Tr cells are not contaminated by potential pathogenic T
cells (such as Th17 cells). Thus, rapamycin can be used to expand in vitro the CD4+CD25+FOXP3+ Tr
cells for cellular therapy in T-cell–mediated diseases, in association with organ transplantation or
bone marrow transplantation, and in the treatment or prevention of GvHD.
The international patent application was published as WO2006090291.
Patent granted in Europe (EP1869163), US (US8562974), Japan (JP5095420) and Australia
(AU2006217546). Patent application pending in Canada.
Relevant Publications
-Battaglia M. Potential T regulatory cell therapy in transplantation: how far have we come and how far can
we go?” Transplantation 2010; 23(8):761-70. Review
-Monti P, Scirpoli M, Maffi P, Piemonti L, Secchi A, Bonifacio E, Roncarolo MG, Battaglia M. Rapamycin
monotherapy in patients with type 1 diabetes modifies CD4+CD25+FOXP3+ regulatory T-cells. Diabetes.
2008 Sep;57(9):2341-7.
-Roncarolo MG, Battaglia M. T regulatory cell immunotherapy for tolerance to self- and allo-antigens in
humans. Nature Reviews Immunology 2007, 7:585-598. Review
-Battaglia M, Stabilini A, Migliavacca B, Horejs-Hoeck J, Kaupper T, Roncarolo MG. Rapamycin promotes
expansion of functional CD4+CD25+FOXP3+ regulatory T cells of both healthy subjects and type 1
diabetic patients. J Immunol. 2006 Dec 15;177(12):8338-47.
-Battaglia M, Stabilini A, Draghici E, Migliavacca B, Gregori S, Bonifacio E, Roncarolo MG. Induction of
Tolerance in Type 1 Diabetes via Both CD4+CD25+ T Regulatory Cells and T Regulatory Type 1 Cells.
Diabetes. 2006 Jun;55(6):1571-80.
-Battaglia M, Stabilini A, Roncarolo MG. Rapamycin selectively expands CD4+CD25+FoxP3+ regulatory
T cells. Blood 2005 Jun 15;105(12):4743-8.
Competitive Advantages
•Rapamycin expands both murine and human CD4+CD25+FOXP3+ Tr cells with suppressive ability in vitro
obtained from peripheral blood or secondary lymphoid organs.
• Murine CD4+CD25+FoxP3+ Tr cells expanded in vitro by rapamycin prevent allograft rejection in vivo.
• The major disadvantage of cellular therapy with in vitro expanded Tr cells is the risk to concomitantly
expand the T effector cells that could be deleterious once transferred in vivo. Indeed, the present
invention also relates to methods of eliminating/reducing CD4+CD25- T effector cells (namely Th17 cells).
• CD4+CD25+ Tr cells may also be able to modulate GVHD whilst preserving the graft versus tumor (GVT)
or graft versus leukemia (GVL) effect.
15
The frequency of CD4+FOXP3+ cells was determined by flow cytometry in human CD4+ T cells
repetitively activated in vitro with (T-rapamycin) or without (T-medium) rapamycin (LEFT).
The CD25 expression levels in T cells expanded without (black line) or with rapamycin (red lines) was
determined by flow cytometry (RIGHT).
One representative experiment out of 5 is shown.
Stage of Development
• In vitro long-term exposure of murine CD4+ T cells to rapamycin induces expansion of the naturally
occurring CD4+CD25+FoxP3+ Tr cells, which retain their suppressive functions in vitro and in vivo.
• The ability of CD4+CD25+FoxP3+ Tr cells expanded in vitro by rapamycin to suppress an immune
response in vivo has been successfully tested in a murine model of allogeneic pancreatic islet
transplantation.
• Treatment of human CD4+ T cells, which includes both T effector cells and CD4+CD25+ Tr cells (5-10%
of the total CD4+ T cells), increases by 20 fold the number of CD4+CD25+FOXP3+ Tr cells. The ability of
rapamycin to selectively expand CD4+CD25+FOXP3+ Tr cells to such levels may be limited to the in vitro
approach.
• A pre-GMP protocol for the expansion of human CD4+CD25+FOXP3+ Tr cells in the presence of
rapamycin has been defined.
• Human rapamycin-expanded Tr cells are not contaminated by Th17 cells and retain their suppressive
activity even upon in vivo transfer in immunodeficient mice.
We seek a commercial partner with a strong pipeline in cellular immunotherapy protocols to
further explore the clinical use of rapamycin-expanded Tr cells for the treatment of T-cell
mediated diseases.
For further information on this project please contact:
Business Contact
Paola Pozzi
Head, Office of Biotechnology Transfer
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 4987
Fax: + 39 02 2643 5264
Email: [email protected]
Scientific Contacts
Prof. M. G. Roncarolo / Dr. Manuela Battaglia
Principal Investigators
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 4875/3945
Fax: +39 02 2643 4668
Email: [email protected]
16
“Tr1-dendritic cells and uses thereof ”
Description of the invention
A large panel of immunosuppressive drugs is now available to prevent acute GvHD and allograft rejection
including steroids, cyclosporin, metotrexate, cyclophosphamide, anti-thymocyte globulin, and anti-CD3
mAb.
While these agents have significantly improved graft outcomes, their use has been associated with
numerous and rather significant toxicities. Moreover, continuous drug administration leads to a sustained
general depression of immune responses. All these effects are due to the non-selective mode of action of
the immunosuppressive drugs.
A valid alternative to immunosuppressive regimens for prevention of GvHD and allograft rejection is the
induction of tolerance to the alloantigens expressed by the recipient or by the graft. This toleranceinduction strategy should selectively target only a small fraction of potentially alloreactive T cells and leave
the remaining T cells of the immune system functionally intact.
Peripheral T-cell tolerance can be induced and maintained by a variety of mechanisms, including deletion,
induction of T-cell hypo-responsiveness, and differentiation of T regulatory (Tr) cells. Tr cells include a
wide variety of cells which all have a unique capacity to inhibit effector T-cell responses.
Addition of IL-10 during dendritic cells (DC) differentiation induces a new subset of tolerogenic Tr1-DC
which can be used to generate anergic Tr1.
Tr1-DC are CD14+CD11c+CD11b+, express CD83, CD80, and CD86, and secrete high levels of IL-10 but
low amounts of IL-12. Importantly, IL-10/IL-12 ratio is maintained upon activation with LPS and IFN-g. Tr1DC are refractory to activation and are potent Tr1 cells inducers in vitro.
An international patent application was published as WO2007131575. Notice of allowance in US.
Patent application pending in Canada.
Gene signature of human Tr1 cells are object of a new patent application (WO2013192215). The
patented technology is available for licensing worldwide.
Relevant Publications
Roncarolo MG, Gregori S, Bacchetta R, Battaglia M. Tr1 cells and the counter-regulation of immunity:
natural mechanisms and therapeutic applications. Curr Top Microbiol Immunol. 2014
Bacchetta R, Lucarelli B, Sartirana C, Gregori S, Lupo Stanghellini MT, Miqueu P, Tomiuk S, HernandezFuentes M, Gianolini ME, Greco R, Bernardi M, Zappone E, Rossini S, Janssen U, Ambrosi A, Salomoni
M, Peccatori J, Ciceri F, Roncarolo MG. Immunological Outcome in Haploidentical-HSC Transplanted
Patients Treated with IL-10-Anergized Donor T Cells. Front Immunol. 2014
Gagliani N, Magnani CF, Huber S, Gianolini ME, Pala M, Licona-Limon P, Guo B, Herbert DR, Bulfone A,
Trentini F, Di Serio C, Bacchetta R, Andreani M, Brockmann L, Gregori S, Flavell RA, Roncarolo MG. Nat
Med. 2013
Gregori S, Tomasoni D, Pacciani V, Scirpoli M, Battaglia M, Magnani CF, Hauben E, Roncarolo MG.
Differentiation of type 1 T regulatory cells (Tr1) by tolerogenic DC-10 requires the IL-10-dependent
ILT4/HLA-G pathway. Blood. 2010.
Pacciani V, Gregori S, Chini L, Corrente S, Chianca M, Moschese V, Rossi P, Roncarolo MG, Angelini F.
Induction of anergic allergen-specific suppressor T cells using tolerogenic dendritic cells derived from
children with allergies to house dust mites. J Allergy Clin Immunol. 2010.
Competitive Advantages
Collectively these data indicate that Tr1-DC are a novel subset of tolerogenic DC that secrete high levels
of IL-10 and low levels of IL-12, and are refractory to activation and maturation in vitro.
Tr1-DC induce anergic T cells in short term cultures; anergic T cells induced by Tr1-DC are regulatory T
cells phenotypically and functionally similar to Tr1 cells.
Tr1-DC display low stimulatory capacity, and, importantly, a single round of stimulation with Tr1-DC is
sufficient to induce Tr1 cells.
Tr1-DC induce anergic T cells in pairs with different HLA disparities which can be used as cellular therapy
to prevent GvHD and organ allograft rejection.
Anergized T cells generated with Tr1-DC are stable and they contain a significant proportion of Tr1 cells.
Furthermore, they are able to suppress Ag-specific primary responses and they are induced by short-term
culture.
17
Potential applications
IL-10 promotes the differentiation of a new subset of tolerogenic dendritic cells (Tr1-DC) which can be
used to generate anergic Tr1 cells with limited in vitro manipulation and suitable for potential clinical use to
restore peripheral tolerance.
The ability of Tr1-DC obtained by the present method to induce anergic allo-antigen specific Tr1 cells was
evaluated. In addition, the potential of Tr1-DC to induce T-cell anergy with limited in vitro manipulation in
haplo-identical and HLA-matched un-related donors was investigated.
Peripheral blood naive CD4+ T cells stimulated with allogeneic Tr1-DC are profoundly anergic and acquire
regulatory function. These T cells are phenotypically and functionally similar to Tr1 cells, since they
secrete high levels of IL-10 and TGF-b and suppress T-cell responses.
A.
Adherence
fraction of PBMC
GM-CSF + IL-4
IL-10
Tr1-DC
7 days
B.
Mature DC
Tr1
Tr1-DC
PBMC
no cytokines Tr1
Tr1
Tr1
10
Tr1
days
Tr1
Tr1
Tr1
Anergy
Mature DC
Tr1
Tr1
PBMC
Tr1
Tr1
Suppression
In vitro differentiation of Tr1 cell lines using Tr1- DC.
A. Tr1-DC are differentiated from CD14+ monocytes by culturing with IL-4 and GM-CSF for 7 days in the
presence of exogenous IL-10. B. Tr1 cell differentiation using Tr1-DC. Total PBMC are stimulated with
allogeneic Tr1-DC at 10:1 ratio for 10 days. The resulting Tr1 cell lines are anergic in response to
mature allogeneic DC, and suppress responses of autologous PBMC activated with mDC.
We seek a potential commercial partner with a strong pipeline in cellular immunotherapy protocols
to further explore Tr1-dendritic cells and uses thereof for the generation of anergic Tr1 cells.
For further information on this project please contact:
Business Contact
Paola Pozzi
Head, Office of Biotechnology Transfer
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 4987
Fax: + 39 02 2643 5264
Email: [email protected]
Scientific Contacts
Prof .M. G. Roncarolo / Dr. S. Gregori/ Dr. R.
Bacchetta
Principal Investigators
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 4669
Email: [email protected]
18
“IGFBP3 and uses thereof”
Background
Gastrointestinal disorders, consisting of gastroparesis, abdominal distension, irritable bowel syndrome
and fecal incontinence, are common in individuals with long-standing Type 1 Diabetes (T1D). The
presence of these gastrointestinal symptoms, known as diabetic enteropathy (DE), significantly reduces
the quality of life and is associated with malnutrition, malabsorbtion, body mass loss and cachexia.
Nevertheless, the pathogenesis of DE is largely unknown.
Herein, the inventors demonstrate that long-standing T1D patients with DE exhibit alterations of the
intestinal mucosa and colonic stem cells (CoSCs) and that a dyad consisting of a circulating enterotrophic
regulating factor and its binding protein (insulin-like growth factor (IGF-I), and its binding protein 3,
IGFBP3) finely controls CoSCs and becomes dysfunctional in DE.
Description of Invention
The present invention described a new potential therapeutic target for individuals with intestinal
disorders, in particular diabetic enteropathy (DE), caused by diabetes mellitus of long duration.
The intestinal epithelium is maintained by intestinal Colonic Stem Cells (CoSCs) and their niche. Whether
systemic factors serve to regulate the homeostasis of colonic epithelium and of CoSCs remains to be
established.
The inventors hypothesize that a circulating “hormonal” dyad controls CoSCs and is disrupted in longstanding Type 1 Diabetes (T1D) patients leading to DE (D’Addio F. et al., Circulating IGF-I and IGFBP3
levels control human colonic stem cell function and are disrupted in diabetic enteropathy. Cell Stem Cell
2015). Indeed, long-standing T1D individuals with severe intestinal symptoms, such as diarrhea,
abdominal pain, and constipation, exhibited morphologic abnormalities of intestinal mucosa and significant
alterations in CoSCs. Proteomic profiling of T1D+DE patient serum revealed altered circulating levels of
insulin-like growth factor 1 (IGF-I) and its binding protein 3 (IGFBP3), with evidences of an increased
hyperglycemia-mediated IGFBP3 hepatic release.
IGF-I acts as a circulating enterotrophic factor that promotes intestinal CoSCs proliferation IGF-IR, while
IGFBP3 can block IGF-I signaling by binding circulating IGF-I and reducing its bioavailability. In addition,
and most importantly, the inventors showed that IGFBP3 can alters CoSCs self-renewal potential and
mucosal morphology in vitro and in a preclinical DE model in vivo, by exerting a TMEM219dependent/caspase 8 and 9-mediated toxic effect on CoSCs, in an IGF-I-independent manner. An
exemplification of the proposed effect of circulating IGF-I and IGFBP3 on CoSCs is reported in Figure 1.
Interestingly, restoration of normoglycemia in patients with long-standing T1D, through a kidney-pancreas
transplantation, normalized circulating IGF-I/IGFBP3 levels and reestablished CoSCs homeostasis,
confirming the direct effect of hyperglycemia on hepatic synthesis and release of IGFBP3 and supporting
the findings regarding the existence of circulating factors that control CoSCs.
In addition, and most importantly, a newly generated ecto-TMEM219 recombinant protein, based on the
extracellular domain of the IGFBP3 receptor (TMEM219), quenches peripheral IGFBP3 and prevents its
binding to endogenous IGFBP3 receptor, TMEM219, abrogating IGFBP3 deleterious effects in vitro on
mucosal epithelium and improving DE in diabetic mice in vivo.
Therefore, the present invention suggest that the inhibition of IGFBP3 could represent a promising novel
target for the diagnosis and/or treatment of T1D patients with intestinal disorders.
The European patent application was filed on June 2015.
19
Figure 1: Schematic attempt to represent the effect of circulating IGF-I and IGFBP3 on CoSCs.
Competitive Advantages:
The present invention relates to a method to diagnose and treat intestinal disorders, in particular diabetic
enteropathy, involving IGFBP3 as key molecule. Inventors envision two major commercial advantages:
1. Diagnostic with the generation of a novel kit, based on a fast point of care test, to sense the presence
and eventually to detect and measure the IGFBP3 levels in urine samples for the diagnosis of
intestinal disorders;
2. Therapeutic with the generation of molecules that block IGFBP3 interaction with IGF1 and with its
receptor, TMEM219, according to the following alternative configurations:
Ø fusion protein TMEM219 (IGFBP3 receptor) extracellular domain - Fc Immunoglobulin
(constant portion);
Ø IGFBP3-blocking antibody;
Ø small molecules;
Ø Oligonucleotides blocking the epatic production of IGFBP3.
We seek a potential commercial partner focused on companion diagnostic and novel therapeutic
approaches for treating intestinal diseases, in particular diabetic enteropathy.
For further information on this project please contact:
Business Contact
Paola Pozzi
Head, Office of Biotechnology Transfer
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 4987
Fax: + 39 02 2643 5264
Email: [email protected]
Scientific Contact
Dr. Paolo Fiorina
Transplantation Medicine Unit
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 3739
Fax: + 39 02 2643 3790
Email: [email protected]
20
“Periangioblasts, adult skeletal muscle stem cells for the
treatment of muscular dystrophies”
For the cell therapy of genetic and acquired diseases of striated muscle, the ideal cell population should
be easily obtained from accessible anatomical sites, expandable in vitro to the large number required for
systemic treatment of primary myopathies and more localized (intra-coronary) treatment of
cardiomyopathies. The cells should be able to reach target muscle in vivo and should be easily
transducible with viral vectors.
Mesoderm stem cells include, beside the canonical hematopoietic and mesenchymal stem cells, a number
of newly described and partially characterized stem/progenitor cells that include:
endothelial progenitor cells (EPC), multipotent adult progenitor cells (MAPC), muscle derived stem cells
(MDCS), side population cells (SP), mesoangioblasts, stem/progenitor cells from muscle endothelium,
sinovia, dermis, and adipose tissue. Currently the phenotypic complexity and the lineage relationships of
these cells is largely unexplored and difference among them are based of specific gene expression and
spectrum of differentiation potential.
Description of Invention
The invention describes the isolation of periangioblasts from biopsies of human and mouse
skeletal muscle.
Periangioblasts are defined as mesoderm progenitors cells derived from a sub-population of blood vessels
pericytes of post-natal skeletal muscle; they display high potential to regenerate skeletal muscle.
In the case of human skeletal muscle, the cells can be expanded in vitro for about 20 population doublings
before undergoing senescence as diploid non tumorigenic cells. When transplanted into dystrophic
immune-incompetent mice they give rise to large numbers of new fibers expressing human dystrophin.
This is the first characterization of a human cell population that fulfils all the criteria for a
successfully cell therapy protocol in Duchenne Muscular Dystrophy.
In addition, the same protocol can be applied to biopsies of mouse and human cardiac muscle.
Although primarily designed for muscular dystrophy, this invention may be exploited also for
acquired disorders of skeletal muscle, such as sphincter lesions, hernias or, together with
biomaterials, surgical ablation of small muscles.
The international patent application was published as WO2007093412.
Patent granted in US (US8071380) and China (ZL2007800056575). Patent pending in Europe.
Potential Applications
Skeletal muscle disorders such as Duchenne and other forms of muscular dystrophy, including but not
limited to limb girdle, facio-scapulo-homeral, myotonic, Emery-Dreyfuss etc. Furthermore inflammatory
myopathies may also be treated with skeletal muscle periangioblasts (Galvez et al, J Cell Biol. 2006;
Dellavallle et al., Nature Cell Biology 2007) ).
Phase I/II clinical trial ongoing. Started in March 2011, at San Raffaele Hospital on paediatric
patients affected by Duchenne Muscular Dystrophy.
21
Competitive Advantages
Periangioblasts can be easily isolated from the very biopsy that is used for diagnosis. A needle biopsy is a
tolerable surgery that can be repeated every few years to further the protocol therapy.
Periangioblasts express some of the proteins that leukocytes use to adhere to and cross the endothelium
and thus can diffuse into the interstitium of skeletal muscle when delivered intra-arterially. This is a distinct
advantage over resident satellite cells that cannot do the same.
Catheter mediated delivery to the subclavia and the iliac arteries allow periangioblasts from skeletal
muscle to reach and colonize muscles essential for motility.
Both normal and dystrophic periangioblasts maintain a diploid karyotype, are not tumorigenic in immune
deficient mice and undergo senescence after approximately 20 population doubligs in vitro. More
importantly, when induced to differentiate in vitro, periangioblasts spontaneously differentiate into skeletal
muscle cells with a frequency up to 40% of the population, an efficiency far superior to any other non
myogenic cell tested so far and second only to resident satellite cells which however cannot be delivered
through the circulation. Although not yet tested in a systematic comparative way, the number of dystrophin
positive muscle fibers produced in vivo by periangioblasts is higher than what reported previously for other
cell types (except resident satellite cells).
We seek a potential commercial partner focused on skeletal muscle disorders to further explore
mammalian post-natal progenitors.
For further information on this project please contact:
Business Contact
Paola Pozzi
Head, Office of Biotechnology Transfer
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 4987
Fax: + 39 02 2643 5264
Email: [email protected]
Scientific Contact
Prof. Giulio Cossu
Division of Regenerative Medicine Stem
Cells and Gene Therapy
San Raffaele Hospital and Scientific Institute
Email: [email protected]
22
“Use of neural stem cells to induce neuroprotection in
inflammatory CNS disorders"
Transplantation of neural stem precursor cells in patients affected by CNS disorders characterized by
chronic inflammation (e.g. multiple sclerosis, brain tumors, ischemic stroke) may have a little therapeutic
impact due to recurrent or persisting inflammation that may target and destroy both CNS-resident as well
as transplanted cells. Scientists at San Raffaele Scientific Institute have been able to describe a novel
immunomodulatory mechanism that further implements the canonical beneficial effect given by
transplanted undifferentiated adult neural stem cells (aNPC) in promoting central nervous system direct
cell replacement by acquiring in vivo terminally differentiated phenotype (Pluchino et al., Nature 2005;
Martino G, Pluchino S. Nat Rev Neurosci. 2006. Review; Pluchino et al., Ann Neurol. 2009).
Upon systemic injection, aNPC are able to exert a neuroprotective effect by inducing in situ
programmed cell death of blood-borne CNS-infiltrating pro-inflammatory Th1, but not antiinflammatory Th2 cells in inflamed CNS perivascular area.
The CNS inflammatory microenvironment dictates aNPCs cell fate and thus their therapeutic efficacy:
when neurodegeneration prevails, transplanted aNPCs acquire a mature phenotype and thus replace
damaged neural cells, while when neuroinflammation predominates, transplanted aNPCs survive to
recurrent inflammatory episodes by retaining both an undifferentiated phenotype and notable proliferating
capacities.
NPC are recruitment at the site of CNS inflammation. In these areas, aNPCs maintain their plasticity
(proliferation vs differentiation), survive over time and exert their neuroprotective effect by inducing in situ
programmed cell death of blood-borne CNS-infiltrating pro-inflammatory Th1 but not anti-inflammatory
Th2 cells. Thus, in vivo aNPC transplantation could be beneficial to induce brain repair and
neuroprotection.
The international patent application was published as WO2007015173. Patent pending in Europe.
Stage of Development
Scientists transplanted subventricular zone (SVZ)-derived syngenic adult NPCs (aNPC) in a mouse model
of chronic-recurrent autoimmune CNS inflammation (relapsing-remitting experimental autoimmune
encephalomyelitis, R-EAE). While assessing their therapeutic potential, they have been able to prove that
inflamed CNS perivascular areas may function during R-EAE as ideal, although atypical, niche-like areas
where transplanted cells can survive for long-term (up to 3 month post transplantation) as “bona fide”
aNPCs. It has also been demonstrated that systematically injected adult syngenic NPCs use constitutively
activated integrins and functional chemokine receptors to selectively enter the inflamed CNS.
Competitive Advantages
-Undifferentiated adult NPCs have relevant therapeutic potential in chronic inflammatory CNS disorders
because they display immune-like functions that promote long-lasting neuroprotection in inflamed CNS
perivascular area
-aNPC-mediated apoptosis of blood-borne CNS-infiltrating encephalitogenic T cells promotes
long lasting neuroprotection in chronic inflammatory CNS disorders.
-Intravenously-injected aNPCs accumulate selectively within CNS inflamed areas using constitutively
functional homing molecules (e.g., a4 integrins and GPCRs) canonically used by pathogenic CNSinfiltrating blood borne lympho/monocytes.
-Once within the CNS, therapeutic (anti-inflammatory) aNPCs maintain preferentially an undifferentiated
phenotype upon transplantation, thus being potentially able to escape from chronic CNS-reactive
autoimmunity.
-It has been demonstrated that considerable numbers of transplanted aNPCs maintain capacity of
proliferation in vivo even 100 days after transplantation, thus being potentially able to modulate their in
vivo fate (proliferation vs quiescence vs migration and differentiation) in response to specific
environmental signals (e.g., cytokines, chemokines, stem cell regulators).
23
Figure. In vitro and in vivo analysis of CD3+ cells undergoing apoptosis. a and b, Spinal cord
perivascular areas stained for TUNEL from either sham- (a) or aNPC-treated R-EAE mice (b, 20X
magnification). Few apoptotic cells (arrows) are visible in a, while the great majority of the cells
surrounding the blood vessel in b are TUNEL+ (black dots). c Spinal cord perivascular area double
stained for TUNEL (dark grey) and CD3 (dark brown) (dashed arrow, TUNEL+CD3- cell; solid arrow,
TUNEL+CD3+; Scale bar, 30 μm). d-g, Representative consecutive (5 μm-tick ) spinal cord sections –
stained for CD3 (brown dots in d and f) or TUNEL (black dots in d and f) – showing perivascular areas
from sham-treated (d and e) or aNPC-injected (f and g) R-EAE mice (40X magnification). Nuclei in d and
f have been counterstained with haematoxilin. The great majority of apoptotic cells expressing CD3 –
which are significantly increased in aNPC-treated mice (p< 0.005 vs. sham-treated) – are confined within
perivascular inflamed CNS areas,as early as 2 weeks p.t. (30 dpi). h, CD3/CD28 activated spleen-derived
lymphocytes undergo apoptosis (AnnexinV+/PI- cells) when co-cultured with aNPCs (single well, black
bars; trans-well, white bars). i, Pro-inflammatory cytokine-conditioned aNPCs express mRNA of proapoptotic molecules. Arbitrary units (AU) represent fold induction of mRNA levels between conditioned
and non-conditioned cells.
We seek a commercial partner with a strong pipeline in Stem Cells, to further the clinical use of
adult neural stem cells in chronic CNS disorders.
For further information on this project please contact:
Business Contact
Paola Pozzi
Head, Office of Biotechnology Transfer
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 4987
Fax: + 39 02 2643 5264
Email: [email protected]
Scientific Contact
Prof. Gianvito Martino, Director
Division of Neuroscience
San Raffaele Hospital and Scientific Institute
Tel: +39 022643 4853
Fax: +39 022643 4855
Email: [email protected]
24
“A non-oxidable HMGB1 mutant for wound healing”
Background, Invention and Potential Applications
High Mobility Group Box 1 (HMGB1) is a nuclear protein that signals tissue damage when released into
the extracellular medium, and thus works as a Damage Associated Molecular Pattern (DAMP) (Bianchi,
2007). Extracellular HMGB1 can act both as a chemoattractant for leukocytes and as a proinflammatory
mediator to induce both recruited leukocytes and resident immune cells to release TNF, IL-1, IL-6 and
other cytokines. Notably, immune cells secrete HMGB1 when activated by infection or tissue damage
(Andersson and Tracey, 2012); mesothelioma and other cancer cells secrete HMGB1 constitutively (Jube
et al., 2012). Recently, our scientists’ results indicated that different molecular forms of HMGB1
orchestrate both key events in sterile inflammation, leukocyte recruitment and activation of cytokine
release.
There is the need to identify HMGB1 variants, that maintain chemoattractant properties but do not
induce cytokine/chemokine production.
To achieve the mutually exclusive process of recruiting inflammatory cells without activating them to a
pro-inflammatory state, our scientists performed studies on the involvement of individual cysteines in the
cytokine-stimulating and chemotactic activities of HMGB1 by generating HMGB1 mutants. The activity of
these HMGB1 variants, has been tested on monocytes/macrophages and fibroblasts and they all failed to
induce cytokines/chemokines expression by macrophages but they all induced fibroblast and monocyte
migration. Namely, they show that by generating a non-oxidizable HMGB1 mutant in which serines
replace all cysteines (i.e. 3S-HMGB1) does not promote cytokine production, but is more effective
than wild-type HMGB1 in recruiting leukocytes in vivo (Venereau et al., JEM 2012) .
Overall, our inventors’ work demonstrates how different redox states of HMGB1 impact its chemotactic
activities leading to therapeutic intervention particularly, in the treatment of a pathology requiring
tissue regeneration, recovery from wounds, fractures and physical trauma, ischemia and recovery
thereof of various tissues and organs.
An international patent application was published as WO2014016417. Patent applications pending
in Europe and US. The patented technology is available for licensing worldwide.
Results and Stage of Development
Our scientists investigated the redox state of HMGB1 in vivo during muscle injury and the subsequent
sterile inflammation, using electrophoretic mobility as an assay. Tibialis anterior muscles of mice were
damaged or not by cardiotoxin (CTX) injection, which causes muscle cell necrosis (Ownby et al., 1993).
Muscles were harvested 2, 6, 24, or 72 h after CTX injection. HMGB1 was barely detectable in the
medium bathing healthy muscles, but was abundant in the medium bathing CTX-injured muscles.
In the model of muscle injury, all-thiol-HMGB1 is prevalent in the extracellular environment immediately
after damage, and disulfide-HMGB1 appears a few hours later; our scientists suggest that all-thiol-HMGB1
is released first to recruit leukocytes, which in turn produce disulfide-HMGB1 directly by secretion and/or
indirectly by partially oxidizing extracellular HMGB1 with ROS (reactive oxygen species). Finally,
sustained ROS production eventually induces the terminal oxidation of HMGB1, which gets inactivated
during the resolution of inflammation. Thus, disulfide-HMGB1 can be considered as a marker of tissue
damage.
Inventors are now testing the molecule in the recovery from muscle damage, with encouraging results and
are discussing the models available for testing the molecule to promote healing of bone fractures, which
they want to address with high priority.
25
25
3S-HMGB1 induces leukocyte recruitment in vivo.
Because 3S-HMGB1 is resistant to oxidation, our scientists hypothesized that its activity in vivo should not
be modified by ROS production. They previously showed (Schiraldi et al., 2012) that the HMGB1–CXCL12
heterocomplex induces a massive influx of leukocytes into air pouches created by the injection of air in the
dorsal derma of mice; such air pouches provide a cavity into which drugs can be administered and from
which recruited cells can be recovered. They injected into air-pouches WT or 3S-HMGB1 (300 pmol)
together with CXCL12 (10 pmol). HMGB1 (WT or 3S) or CXCL12 alone failed to induce leukocyte
recruitment, but both WT and 3S-HMGB1 in association with CXCL12 induced a massive influx of
leukocytes (Fig. 5 B). Notably, the number of recruited leukocytes was increased in response to 3SHMGB1–CXCL12 compared with WT HMGB1–CXCL12.
At day 6, the air pouches were injected with 200 μl of PBS containing 10 pmol CXCL12, 300 pmol HMGB1
(WT or 3S), or both.
Fig. B: scientists injected into air-pouches WT or 3S-HMGB1 (300 pmol) together with CXCL12 (10 pmol).
HMGB1 (WT or 3S) or CXCL12 alone failed to induce leukocyte recruitment, but both WT and 3S-HMGB1
in association with CXCL12 induced a massive influx of leukocytes. Notably, the number of recruited
leukocytes was increased in response to 3S-HMGB1–CXCL12 compared with WT HMGB1–CXCL12.
Fig. D: same experiment have been performed in the presence or not of an antioxidant such as Nacetylcysteine (NAC). After 6 h, cells were collected from the air pouches, stained with anti-Ly6C and antiCD11b antibodies, and analyzed by flow cytometry (WBCs, white blood cells; *, P < 0.05; **, P < 0.01; ***,
P < 0.001, ANOVA plus Dunnett’s posttest).
Competitive Advantages
Importantly, the invention is a valuable tool to be used for therapeutic applications in order to promote cell
recruitment for repairing damaged tissue.
We seek a potential commercial partner focused on developing selected HMGB1 variants to be
exploited as therapeutic tool.
For further information on this project please contact:
Business Contact
Paola Pozzi
Head, Office of Biotechnology Transfer
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 4987
Fax: + 39 02 2643 5264
Email: [email protected]
Scientific Contact
Prof. Marco E. Bianchi
Division of Genetics and Cell Biology
San Raffaele Hospital and Scientific Institute
Tel: +39 02 26434765
Fax: + 39 02 2643 5544
Email: [email protected]
26
“MicroRNA-regulated Viral Vectors”
MicroRNAs are a family of small, non-coding RNAs involved in downregulating gene expression by
recognizing in a sequence-specific manner target mRNAs.
The present invention describes a gene transfer vector system that utilize the microRNA posttranscriptional gene silencing machinery for regulating transgene expression. Lentiviral vectors for
transgene expression for gene therapy can be engineered with microRNAs target sequence in order to be
recognized by endogenous microRNAs cell type specific. Thus, regulation of transgene expression in a
subset of cells can be achieved. Moreover, a combinations of miRNA target sequences can be used to
obtain vectors with highly specific cell expression patterns.
This invention could be employed to prevent immune-mediated rejection of the transferred gene.
Indeed, as an example, the inventors have demonstrated that transgene expression from a ubiquitously
expressed promoter can be prevented precisely in a hematopoietic cell line by using a vector that displays
miR-142 target sequence at the transgene’s 3’UTR, as shown in the figure below: in those cells in which
miR-142 is being expressed, miR-142 specifically recognize its target sequence and therefore it inhibits
transgene expression; indeed, miR-142 has a cell-type specific expression pattern in hematopoietic
tissues. Thus in this example, the system does not reduce transgene expression in other cell types.
Upon vector administration in vivo, this will avoid transgene
expression in antigen presenting cells (APC) of the immune
system, which are part of the hematopoietic system, and
thereby prevent the initiation of an immune response
against the transgene. Conceivably, when applied to a
tissue-specific promoter which targets expression to
hepatocytes, it would allow suppressing any ectopic
expression in transduced APC. This would potentially
solve a major hurdle and long-standing problem in gene
transfer; namely, immune-mediated rejection of the
transferred gene (Brown et al., Nat Med. 2006; Brown et
al., Blood. 2007).
The international patent application was published as WO2007000668.
Patent pending in India, Singapore, Israele, US, Japan and China. Patent granted in Canada, S.
Korea (10-1373548). Intention to grant in Europe. Available for out-licensing in specific fields only.
Relevant Publications
-Matsui H et al., A microRNA-regulated and GP64-pseudotyped lentiviral vector mediates stable
expression of FVIII in a murine model of Hemophilia A. Mol Ther. 2011
-Matrai J et al., Hepatocyte-targeted expression by integrase-defective lentiviral vectors induces antigenspecific tolerance in mice with low genotoxic risk. Hepatology. 2011
-Naldini L. Ex vivo gene transfer and correction for cell-based therapies. Nat Rev Genet. 2011
-Gentner B et al., Identification of hematopoietic stem cell-specific miRNAs enables gene therapy of
globoid cell leukodystrophy. Sci Transl Med. 2010
-Sachdeva R et al., Tracking differentiating neural progenitors in pluripotent cultures using microRNAregulated lentiviral vectors. Proc Natl Acad Sci U S A. 2010
-Annoni A et al., In vivo delivery of a microRNA-regulated transgene induces antigen-specific regulatory T
cells and promotes immunologic tolerance. Blood. 2009
-Brown BD, Naldini L. Exploiting and antagonizing microRNA regulation for therapeutic and experimental
applications. Nat Rev Genet. 2009
-Brown BD et al., Endogenous microRNA can be broadly exploited to regulate transgene expression
according to tissue, lineage and differentiation state. Nat Biotechnol. 2007
-Brown BD et al., A microRNA-regulated lentiviral vector mediates stable correction of hemophilia B mice.
Blood. 2007
-Brown BD et al., Endogenous microRNA regulation suppresses transgene expression in hematopoietic
lineages and enables stable gene transfer. Nat Med. 2006
27
Potential Applications
Current vector transcription control approaches mostly rely on the delivery of enhancer-promoter elements
taken from endogenous genes. Using these approaches, reconstitution of highly specific gene expression
patterns, as often required for gene transfer and therapy applications, is limited by the delivery system, the
vector capacity, and the positional effects of insertion (for integrating vectors). By developing new vectors
which take advantage of endogenously expressed microRNAs for their regulation, the inventors have
added a layer of control to the vectors that did not previously exist.
This new approach allows specific repression of gene expression in selected cell types and lineages.
Furthermore, vectors with highly specific cell expression pattern may be useful in screening protocols as
research tool.
Stage of Development
In vivo studies have been performed to validate these concepts.
LV.PGK.GFP.WPRE is a lentiviral vector in which transgene expression is controlled by the ubiquitously
expressed PGK Promoter.
.
By adding microRNA target sequences (<23bp) to the transgene’s 3’UTR, which are targeted by tissue
specific miRNA, we can create a vector with tissue-restricted expression.
Moreover, we can add combinations of miRNA target sequences to obtain vectors with highly specific cell
expression patterns.
In addition, the inventors are carrying out studies to determine if this invention enables establishment of
long-term transgene expression, and therapeutic activity, after systemic gene transfer in the absence of an
immune response. If so, this invention would constitute an important component of any vector system
intended for systemic delivery of a therapeutic transgene.
Competitive Advantages
With this system we can reach much more stringent control of transgene exprssion than is current
possible with existing technologies.
When applied to integrating vectors, it can circumvent problems of transgene dysregulation, which can
occur as a result of insertional position effects (integration next to strong promoter/enhancer sequences
that override the transcriptional control of the vector-internal promoter) and enable highly cell-specific
patterns of transgene expression.
We seek a potential commercial partner with a strong pipeline either in human gene therapy
interested in avoiding immune mediated rejection of the transgene or/and in screening
procedures.
For further information on this project please contact:
Business Contact
Paola Pozzi
Head, Office of Biotechnology Transfer
San Raffaele Hospital and Scientific
Institute
Tel: +39 02 2643 4987
Fax: + 39 02 2643 5264
Email: [email protected]
Scientific Contact
Prof. Luigi Naldini
Director San Raffaele Telethon Institute for Gene
Therapy
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 4681
Fax: +39 02 2643 4621
Email: [email protected]
28
“New promoters and new lentiviral vectors:
efficient and coordinated expression of multiple genes”
The present invention describes new promoters designed and constructed for multiple gene expression
that have been incorporated into state-of-the-art, self-inactivating lentiviral vectors to reach stable
integration and efficient, coordinated expression in all transduced cells.
The cassette includes a minimal promoter joined upstream to an efficient promoter in the opposite
orientation. The rationale behind this design is the sharing of orientation-independent enhancer activity
contributed from the efficient promoter between the two closely linked basal promoters acting in opposite
directions. The bi-directional promoter then mediates the coordinated, divergent transcription of
two mRNAs (Amendola M., Venneri MA., Biffi A, Vigna E., Naldini L., “Coordinate dual-gene transgenesis
by lentiviral vectors carrying synthetic bidirectional promoters” Nat. Biotechnol. 2005).
The international patent application was published as WO2004094642.
Patents granted in Europe (EP1616012), US (US8501464 ) and Canada (CA2523138). Available for
outlicensing in specific fields.
Comparing Bidirectional and Bicistronic LV
DNGFR
36%
Mean=2276
16.4%
Mean=1200
GFP
DNGFR
Mean=164
Mean=49.5
GFP
Efficient and coordinated gene expression in human hematopoietic progenitor cells by bidirectional
lentiviral vectors. CD34+ hematopoietic progenitors were purified from cord blood and transduced with
normalized amounts of the indicated vectors (5x107 TU/ml) expressing a truncated form of the human lowaffinity NGF receptor (DLNGFR) and GFP (Green Fluorescent Protein). The bidirectional MA-1 vector was
compared to the best performing IRES bicistronic vector. The bidirectional vector reached a higher
frequency of DLNGFR+ cells that also displayed a higher expression level and this was even more true for
GFP. In the left panels the dot plot analysis for both DNGFR and GFP expression are shown; in the center
panels the histograms for DNGFR expression are shown; in the right panels the histograms for GFP
expression of the DNGFR expressing cells are shown.
Stage of Development
Murine transplantation studies are in evaluation, to verify the advantages of such vectors for the
amplification or selection of polyclonal population of engineered hematopoietic stem cells
We are also performing vector transgenesis experiments to prove efficient and coordinated expression of
two exogenous genes in all types of tissues
29
Potential Applications
Expression of two or more exogenous genes in an efficient and coordinated manner within the same cell is
a difficult but important task to reach. Our system allows the coordinated expression of two or more genes
and can find several applications in:
•Gene function and in vitro and in vivo target validation studies
•Gene therapy
•Expression of multiple genes in animal cells
•Generation of transgenic animals and\or knock down of multiple genes
•Manufacturing of medicaments
Competitive Advantage
Our system compared with the use of two separate expression vectors, use of two expression
cassettes driven by different promoters in a single vector, or with IRES bicistronic vectors shows a:
Ø
More efficient expression
Ø
Coordinated expression of both genes in virtually all transduced cells
Ø
Efficient integration and robust expression by lentiviral vector delivery
Ø
Cell type independent application
We seek a potential commercial partner with a strong pipeline in human gene therapy for the
treatment of genetic and acquired diseases or should be a biotech company selling research
reagents for creation of animal models and for in vitro target validation.
For further information on this project please contact:
Business Contact
Paola Pozzi
Head, Office of Biotechnology Transfer
San Raffaele Hospital and Scientific
Institute
Tel: +39 02 2643 4987
Fax: + 39 02 2643 5264
Email: [email protected]
Scientific Contact
Prof. Luigi Naldini
Director San Raffaele Telethon Institute for
Gene Therapy
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 4681
Fax: +39 02 2643 4621
Email: [email protected]
30
“A method for the ex vivo production of fully functional genemodified human T lymphocytes ”
The success of T-cell gene therapy depends on the ability to produce high numbers of clinical-grade genemodified T lymphocytes ex vivo without impairing their functionality. In allogeneic hemopoietic cell
transplantation (allo-HCT), donor T cells can be modified to express a suicide gene, i.e. a gene that
encodes for a factor able to transform a non-toxic prodrug into a toxic compound, thus enabling their
selective elimination. This allows for the exploitation of donor T-cell alloreactivity for a therapeutic graftversus-leukemia (GvL) effect, while providing a safety switch of graft-versus-host disease (GvHD).
In the setting of suicide gene therapy applied to allo-HCT, the preservation of gene-modified T-cell
functionality coincides with the preservation of their alloreactivity. To date, this is an unmet need in
biomedicine, since current clinical protocols for the production of gene-modified T cells with viral vectors
reduces their alloreactivity.
The international patent application was published as WO2007017915.
Patent granted in Europe (EP1956080), US (US8999715) and divisional patent application pending
in US.
Description of Invention
Scientists at San Raffaefe Scientific Institute have identified two essential requisites for the ex vivo
production of fully alloreactive gene-modified human T lymphocytes:
i) the activation with anti-CD3 and anti-CD28 monoclonal antibodies coupled to cell-sized paramagnetic
beads (CD3/CD28-beads) prior to genetic modification with viral vectors (reported in Bondanza et al,
Blood 2006)
ii) the culture of gene-modified T cells with a combination of IL-7 and IL-15 (reported in Kaneko et al,
Blood 2009).
Figure 1. IL7/IL-15 are required for the ex vivo production of
human T lymphocytes modified with viral vectors to express
the prototypic suicide gene TK after CD3/CD28-bead
activation
Figure 2. Human T lymphocytes modified with TK
after CD3/CD28-bead activation are as alloreactive as
unmodified cells in a fully humanized animal model
of GvHD based on the grafting of HLA-mismated
human skin onto NOD/scid mice
Moreover, the use of the T central memory (TCM) phenotype has been identiified as a promising candidate
biomarker for the detection of fully alloreactive gene-modified human T cells.
31
Stage of Development
Scientists have established a protocol for the ex vivo production gene-modified human T lymphocytes
(Figure 1) and demonstrated the full preservation of their alloreactivity in a fully humanized mouse model
of GvHD (Figure 2). Currently, the functionality of gene-modified T cells produced with this protocol is
under test in the setting of the adoptive T-cell gene therapy of leukemia and other tumors using TCR and/
or chimeric antigen receptors specific for tumor- associated antigens
We seek a commercial partner with a strong expertise in cancer adoptive immunotherapy
strategies to further explore advantages of the present method in clinical applications.
For further information on this project please contact:
Business Contact
Paola Pozzi
Head, Office of Biotechnology Transfer
San Raffaele Hospital and Scientific
Institute
Tel: +39 02 2643 4987
Fax: + 39 02 2643 5264
Email: [email protected]
Scientific Contacts
Prof. Maria Chiara Bonini / Dr. Attilio Bondanza
Principal Investigators
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 4790
Fax: +39 02 2643 4786
Email:[email protected];
[email protected]
32
“New gene therapy approach to induce antigen-specific
immunological tolerance”
In vivo induction of antigen-specific Treg has been a long-sought goal of experimental medicine. Here we
report in vivo induction of antigen-specific Treg and active immune tolerance against foreign antigens by
hepatocyte-targeted Integrase-Deficient Lentiviral Vectors (IDLV). This platform enables efficient liver gene
transfer for a window of time and induces immune tolerance to the encoded antigen in a “hit and run”
approach, without the need for long-term integration, thus providing enhanced safety as compared to
Integrase-Competent Lentiviral Vectors (ICLV). Hepatocyte-targeted IDLV are promising new vectors for a
broad range of applications, and primarily for
1) induction of antigen-specific Tregs in “inverse vaccination” strategies to
1a) tolerize individuals to protein replacement therapies (such as in lysosomal storage disorders and
hemophilias or other plasma protein deficiencies). One of the major complications of protein
replacement therapies is the development of neutralizing antibodies against the therapeutic protein.
Thus patients undergoing these therapies would strongly benefit from the induction of tolerance to
the therapeutic protein.
1b) prevent or revert the development of autoimmune diseases (such as multiple sclerosis,
diabetes) and allergic diseases.
2) reversible hepatic gene transfer of therapeutic proteins of therapeutic proteins, such as interferon (IFN)
and other cytokines, in chronic viral hepatitis or hepatic tumors; gene-based delivery may provide therapeutic concentrations of the factor at the disease site with limited systemic exposure and only for a defined
window of time.
The international patent application was published as WO2010055413.
Patent pending in US. Intention to grant in Europe.
The present invention describes a new strategy for inducing antigen-specific immunological
tolerance which exploits endogenous miR-142 regulation in combination with transient liver gene
delivery with non integrating vectors (IDLV).
1
2
Figure 1. GFP-expressing IDLV.ET.GFP.142T (grey
bars) vs. ICLV.ET.GFP.142T (black bars) injected
intravenously to adult mice (n=20 for IDLV mice and n=4
for ICLV mice in three independent experiments) and
measured GFP expression and vector DNA contents in
the liver at different times post-injection.
Figure 2. Transgene-specific tolerance after IDLV liver
gene transfer. Quantification of the CD8+ GFP200-208pentamer-positive T cells infiltrating the liver of mice
treated with the indicated IDLV (IDLV.PGK n=9;
IDLV.PGK.142T n=6; IDLV.ET.142T n=3) after antigen
re-challenge (by intramuscular vaccination with GFPencoding plasmids) 6 weeks after IDLV treatment.
3
Figure 3. Induction of transgene-specific Tregs by IDLV
treatment CD4+ cells isolated from OTII Ly5.2 Foxp3GFP transgenic mice were FACS-sorted to remove
GFP+ cells obtaining an homogeneous population of
CD4+ non-regulatory T cells with a unique antigen
specificity (OVA323-339 presented in IAb molecule). (A)
Tregs-depleted OTII CD4+GFP- (2.5&times;106/mouse)
were adoptively transfer intravenously into naive
C57BL/6 Ly5.1 recipient mice one day before the
injection of IDLV.PGK (n=3) or IDLV.ET.142T (n=3)
encoding for OVA. Three weeks after IDLV
administration livers were harvested and infiltrating
lymphocytes isolated. OVA-specific induced Tregs were
measured as GFP+ cells gated on CD4+Ly5.2+. (B) a
representative histogram and (C) mean % induced
Tregs +/- SEM is reported.
33
Competitive Advantages
These results demonstrate a new strategy of tolerance induction which exploits miR-142-regulation in
combination with transient gene delivery. This approach renders treated mice unresponsiveness to
vaccination against strong neoantigens including a therapeutic protein (factor IX in hemophilia B mice) at 6
weeks after IDLV delivery (Fig. 2) and induces the conversion of naïve into antigen-specific Tregs in the liver
as assessed at three weeks after IDLV delivery (Fig. 3). This strategy allows inducing long-lasting tolerance
to transgene-encoded antigens without the need for long-term transgene expression and reducing risks
associated with vector integration.
Stage of Development
In Vivo proof of principle has been achieved with different model antigens (GFP, ovalbumin) and a
therapeutic protein (coagulation factor IX in hemophilia B mice).
Further validation for more specific therapeutic application of this technology are ongoing.
Relevant Publications
Annoni A, Cantore A, Della Valle P, Goudy K, Akbarpour M, Russo F, Bartolaccini S, D'Angelo A, Roncarolo
MG, Naldini L. Liver gene therapy by lentiviral vectors reverses anti-factor IX pre-existing immunity in
haemophilic mice. EMBO Mol Med. 2013
Cantore A, Nair N, Della Valle P, Di Matteo M, Màtrai J, Sanvito F, Brombin C, Di Serio C, D'Angelo A,
Chuah M, Naldini L, Vandendriessche T. Hyperfunctional coagulation factor IX improves the efficacy of gene
therapy in hemophilic mice. Blood. 2012
Mátrai J, Cantore A, Bartholomae CC, Annoni A, Wang W, Acosta-Sanchez A, Samara-Kuko E, De Waele L,
Ma L, Genovese P, Damo M, Arens A, Goudy K, Nichols TC, von Kalle C, L Chuah MK, Roncarolo MG,
Schmidt M, Vandendriessche T, Naldini L. Hepatocyte-targeted expression by integrase-defective lentiviral
vectors induces antigen-specific tolerance in mice with low genotoxic risk. Hepatology. 2011.
Annoni A, Brown BD, Cantore A, Sergi LS, Naldini L, Roncarolo MG. In vivo delivery of a microRNAregulated transgene induces antigen-specific regulatory T cells and promotes immunologic tolerance. Blood.
2009.
Brown BD, Naldini L. Exploiting and antagonizing microRNA regulation for therapeutic and experimental
applications. Nat Rev Genet. 2009 Review.
We seek an industrial partner focused on protein replacement therapies and/or treatment of
autoimmune disorder to further develop this technology for clinical applications.
For further information on this project please contact:
Business Contact
Paola Pozzi
Head, Office of Biotechnology Transfer
San Raffaele Hospital and Scientific
Institute
Tel: +39 02 2643 4987
Fax: + 39 02 2643 5264
Email: [email protected]
Scientific Contacts
Prof. Luigi Naldini
Director San Raffaele Telethon Institute for Gene
Therapy
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 4681
Fax: +39 02 2643 4621
Email: [email protected]
34
“Myeloid Microvesicles are a marker and
therapeutic target for neuroinflammation”
Microvesicles (MVs) have been indicated as important mediators of intercellular communication and are
emerging as new biomarkers of tissue damage.
MVs released by microglia/macrophages in vivo were detected in cerebrospinal fluis (CSF) of healthy
controls. In relapsing and remitting experimental autoimmune encephalomyelitis (EAE) mice, the
concentration of myeloid MVs in the CSF was significantly increased and closely associated to disease
course. Analysis of MVs in the CSF of 28 relapsing patients and 28 patients with clinical isolated syndrome
from two independent cohorts, revealed higher levels of myeloid MVs than in 13 matched-age controls,
indicating a clinical value of MVs as companion tool for disease to capture disease activity
diagnosis. Myeloid MVs were found to spread inflammatory signals both in vitro and in vivo, at the site of
administration, while mice impaired in MV shedding were protected from EAE, suggesting a pathogenic
role for MVs in the disease. Interestingly, FTY720 (a specific inhibitor of A-SMase, the enzyme that
controls MV production, and the first approved oral MS drug), significantly reduced the amount of MVs in
the CSF of EAE treated mice.
These findings identify myeloid MVs as a valuable marker and therapeutic target of brain
inflammation (Verderio et al., Annals of Neurology 2012).
The international patent application was published as WO2011107962. Patent granted in US
(US8999655).
Description of Invention
To verify whether the findings obtained in the mouse model can be extended to humans, we collected CSF
from two independent cohorts of healthy donors, patients with Clinically Isolated Syndrome (CIS), patients
with definite primary progressive or relapsing-remitting multiple sclerosis (PPMS and RRMS, respectively),
the latter during a stable phase of the disease (stable RRMS), or during an acute attack (acute RRMS)
and patients from other neurologic diseases.
These data indicate CSF MVs as novel exploratory biomarker of microglia/macrophage activation.
Myeloid MVs were significantly increased in the CSF from both cohorts of CIS and relapsing RRMS
patients (Figure below).
Furthermore, the pathogenic role of MVs in the inflammatory response was demonstrated in vivo by
showing that injection of microglia-derived MVs induces the formation of inflammatory foci at the site of
delivery.
35
Potential Applications
CSF myeloid MVs as novel exploratory biomarkers of microglia/macrophage activation in vivo.
In MS, the most common neuroinflammatory disease, CSF MVs may be useful as companion tool for
to monitor disease diagnosis activity and for monitoring the efficacy of drugs, or to identify very
active patients likely to need a prompt shift to second line treatments or CIS patients needing early
treatment.
Microglia activation, however, is also associated to several other CNS diseases, like, for example,
neuromyelitis optica, or brain tumors, that in fact display increased CSF MVs. Thus CSF MVs monitoring
may provide valuable information in several different neurological disorders.
We also propose that MVs produced by microglia/macrophages and leaking into the CSF may represent a
rich source of information on microglia/macrophage activation in the brain, which may lead to the
identification of specific disease cell signature through the analysis of their content.
Competitive Advantages
The concentration of microglia/macrophage-derived MVs in mouse CSF reflects the course and severity of
EAE. Consistently, the amount of MVs in human CSF is higher in patients presenting with the first clinical
symptom of MS or in relapsing patients as compared to patients in a stable phase of the disease or
healthy controls.
Given MVs are a unique way for exchanging integrated signals, targeting MVs may represent a
therapeutic strategy more advantageous than classical approaches aimed at neutralizing single
inflammatory molecules in MS.
We seek a potential commercial partner focused on companion diagnostic and novel therapeutic
approaches for treating neuroinflammation in neurological disorders.
For further information on this project please contact:
Business Contact
Paola Pozzi
Head, Office of Biotechnology Transfer
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 4987
Fax: + 39 02 2643 5264
Email: [email protected]
Scientific Contact
Dr. Roberto Furlan
Clinical neuroimmunology
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 2791
Fax: + 39 02 2643 4855
Email: [email protected]
36
“PTGDS pathway activators and uses thereof”
Background
Peripheral neuropathies are an important cause of disability with notable social costs. Peripheral
neuropathies can be primarily demyelinating or axonal neuropathies, although at later stages both
components are affected. Among all peripheral neuropathies, Charcot Marie Tooth neuropathies are rare,
monogenic disorders, characterized by progressive motor and sensory deficits. Much is known of the
genetic causes and clinical aspects of these hereditary neuropathies, but no effective treatment is
available.
Herein, the inventors describe an unknown pathway involved in Peripheral Nervosus System (PNS)
myelin formation and maintenance. Specifically, they demonstrate that in the PNS the intracellular
cleavage of Neuregulin 1 (NRG1) type III and its nuclear translocation induce the expression of the
prostaglandin D2 synthase (PTGDS), which, together with the G-protein-coupled receptor Gpr44,
contributes to myelin formation and maintenance. Thus, modulation of the PTGDS‐Gpr44 signaling axis
could represent a novel approach for the treatment of peripheral demyelinating neuropathies.
Description of Invention
The present invention relates to an activator of the PTGDS pathway for use in the treatment and/or
prevention of a pathology characterized by an altered myelination in the peripheral nervous
system. In particular the pathology is a peripheral neuropathy such as hereditary neuropathy,
inflammatory neuropathy or toxic neuropathy.
While prostaglandins and their synthases have been investigated in the pathogenesis of allergic asthma
and other inflammatory disorders, very few studies have focused their attention on the role of these
molecules in the formation and modulation of Peripheral Nervosus System (PNS) myelin.
Myelin, the insulating organelle enwrapping the axon in central nervous system (CNS) and PNS, is formed
by oligodendrocytes in the CNS and Schwann Cells in the PNS and is essential for rapid conduction of
electrical impulses and neuronal survival. In the PNS, levels of axonal Neuregulin 1 (NRG1) type III, a
member of the Neuregulin family of growth factors, controls all the aspects linked to Schwann cell
development and myelin formation. NGR1 type III, similarly to other growth factors, is processed in the
extracellular region by secretases, namely the beta secretase BACE-1 and the alpha secretase TACE.
In the PNS, NRG1 type III is processed also intramembrane by the γ-secretase complex through a
mechanism regulated by the ErbB receptors. The inventors found that the NRG1 generated fragment
specifically upregulates the Prostaglandin D2 Synthase (PTGDS) gene. Neuronal PTGDS is secreted and
produces the PGD2 prostanoids, a ligand of Gpr44 receptor. Activation of this G protein coupled receptor,
in Schwann cells, leads to dephosphorylation and consequent nuclear translocation of the transcription
factor NFATc4, a triggering event for the expression of key genes (e.g. MBP, MPZ) involved in
myelination (Trimarco A. et al., Prostaglandin D2 synthase/GPR44: a signaling axis in PNS myelination.
Nature Neuroscience 2014). An exemplification of the proposed model for PTGDS/Gpr44 signaling axis
in PNS myelination is reported in Figure 1.
Consistent with these evidences, specific inhibition of PTGDS enzymatic activity impaired in vitro
myelination and caused myelin damage. Accordingly, sciatic nerves of Ptgds- /- transgenic mice are
hypomyelinated. Furthermore, in vivo ablation and in vitro knockdown of glial Gpr44 impaired myelination.
Thus, the authors propose that in the PNS, specific up-regulation of PTGDS expression, following NRG1
type III intracellular cleavage and nuclear translocation, participates in myelin formation and maintenance,
identifying a novel pathway whose modulation could be beneficial for the treatment of peripheral
demyelinating neuropathies. This is the first study implicating prostaglandins as active controller of
myelination. It is therefore an object of the invention an activator of the PTGDS pathway for use in the
treatment and/or prevention of a pathology characterized by an altered myelination in the PNS.
The international patent application was published as WO2015000921.
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Figure 1: NRG1, upon cleavage, translocates into the nucleus to specifically activate PTGDS mRNA expression.
PTGDS protein is then released in the media where it is enzymatically active. PGD2, a PTGDS metabolite, binds to
and activate glial GPR44 that results in dephosphorylation and nuclear translocation of transcription factor NFATc4,
to promote myelin gene expression.
Competitive Advantages:
The present invention relates to a method to treat and possibly diagnose peripheral demyelinating
neuropathies, involving PTGDS and Gpr44, as previously unknown components of an axo-glial
interaction that controls PNS myelination. Inventors envision two major commercial advantages:
1. Diagnostic, with the generation of a method for detecting the presence and/or measuring the amount
of PTGDS and/or of at least one of PTGDS metabolite receptor in a biological sample;
2. Therapeutic, with the generation of an activator PTGDS pathway according to one the following
alternatives:
.
• an activator of PTGDS, such as an activator of PTGDS mRNA expression, an analog of
PTGDS, an antibody that specifically activates PTGDS. Yet preferably the activator of the
PTGDS pathway is obtainable by means of a gene therapy approach. Still preferably the
activator of PTGDS is a fragment of NRG1 type III;
• an agonist of a PTGDS metabolite receptor;
• an activator of PGD2 and/or PGJ2 (PTGDS metabolites) production or;
• any combination thereof.
We seek a potential commercial partner focused on companion diagnostic and novel therapeutic
approaches for treating peripheral demyelinating neuropathies.
For further information on this project please contact:
Business Contact
Paola Pozzi
Head, Office of Biotechnology Transfer
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 4987
Fax: + 39 02 2643 5264
Email: [email protected]
Scientific Contact
Dr. Carla Taveggia
Axo-glial interactions Unit
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 4439
Fax: + 39 02 2643 6164
Email: [email protected]
38
“Composite Scaffold for Tissue Repair”
Orthopaedic surgeons in collaboration with engineers have developed a biphasic scaffold for the
regeneration of the osteochondral tissue, composed of collagen 1 and hydroxyapatite. This scaffold
can be used as a cell-free implant or in combination with a source of cells, it has good press-fit properties
facilitating the filling of the defect and it is stable throughout long term regenerative applications.
The international patent application was published as WO2014184391.
Competitive Advantages
This biphasic scaffold was developed for the treatment and regeneration of osteochondral defects and
presents competitive aspects with respect to the current commercial solutions, in particular:
•A interconnection zone between the upper collagen 1 layer (that represents the chondral phase) and
the lower hydroxyapatite layer (that represents the bony phase), thus conferring a strong integration
of the two materials and allowing for a better stability and integrity of the scaffold;
•An external thin layer made of collagen 1 that surrounds the whole biphasic scaffold, thus conferring
good press-fit qualities to the composite and facilitating the insertion of the scaffold into the
defect;
•A cell free application, as it can be efficiently repopulated by cells deriving from the bone marrow or the
surrounding tissues, as demonstrated by the in vivo experiments in the swine model;
•An early regenerative potential of the chondral tissue, observed after three months in the swine
model
Fig. 1
Untreated
Cell-free scaffold
Cell-seeded scaffold
Figure 1 shows glycosaminoglycans staining of the repaired tissue after 3 months.
As indicated by arrows, the cell free scaffold of the present invention promoted a better regeneration of
the chondral tissue. *** indicates the center of the defect
Fig. 2
Figure 2: arrows show a detail of
the integration between the
collagen and the hydroxyapatite
layers (black arrows)
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Stage of Development
This biphasic scaffold is at a pre-clinical stage of development.
It has been validated in the swine model, in osteochondral defects of the trochlea where the scaffold was
implanted in the unseeded form and in association with autologous chondrocytes.
Inventors are now validating the scaffold by implanting it in the medial femoral condyle (MFC) of the
sheep model, in order to evaluate its potentialities in defects subjected to higher load-bearing activity.
Moreover, a growth factor releasing version of the scaffold will be produced and tested in vivo in order to
potentiate the regenerative qualities. Additionally, a novel version of the scaffold will be tested in MFC of
the sheep model, presenting columnar organization of the hydroxyapatite within the bony part of the
osteochondral substitute, in order to allow a more efficient penetration of the bone marrow mesenchymal
stem cells throughout the entire thickness of the scaffold, to the bony and chondral part. This new version
presents also an important feature: the possibility to be cut and shaped before the implantation, based on
the shape of the defect. A longer time point is planned in order to verify the long term efficacy of its
reparative properties in vivo .
We seek a commercial partner focused on manufacture and distribution of orthopaedic medical
devices intended for regenerative medicine.
For further information on this project please contact:
Business Contact
Paola Pozzi
Head, Office of Biotechnology Transfer
San Raffaele Hospital and Scientific
Institute
Tel: +39 02 2643 4987
Fax: + 39 02 2643 5264
Email: [email protected]
Scientific Contact
Prof. Giuseppe Peretti
Laboratory of Tissue Engineering and
Biomaterials
San Raffaele Hospital and Scientific Institute
Tel: +39 02 5031 9967
Fax: +39 02 6621 4736
Email: [email protected]
40
“Optical platform for ion channel drug screening”
We propose an optical platform based on a proprietary method, that is aimed to screen candidate
compounds acting on ion channels. The proposed method allows to derive experimentally a doseresponse curve concerning specific agonist-receptor interactions by a fully automated procedure
that fits the requirements of high throughput screening and offers a competitive alternative to traditional
electrophysiological techniques.
The international patent application was published as WO2011009825.
Patent granted in Europe (EP2457088). Patent pending in US.
Briefly, fast-VSD-loaded cells are exposed to electric field stimulation in order to promote an amplification
of the ionic currents flowing through the ion channels/transporters in a conductance dependent fashion.
Since conductance values are measured under control conditions and after exposure to specific
treatments, it is possible to estimate the effect of pharmacological treatments on membrane permeability.
A dedicated platform has been designed to exploit the proprietary method, and makes it now possible to
obtain the pharmacological profile of molecules acting on ion channels/transporters in a fully automated
way.
The method can be applied to evaluate:
- direct or indirect activation, modulation or blockade of ion channels or transporters expressed on the
plasma membrane, with the possibility to derive experimentally a concentration-response relationship;
- the direct permeabilizing effect of molecules with channel or transport characteristics (i.e. antibiotics and
antimycotics);
- changes in the basal conductance of the plasma membrane as a marker of the different physiological
states of a cell;
- the cellular and sub-cellular localization of permeability events in identified domains (even in complex
and heterogeneous cellular models).
Stage of Development
The whole process is fully automated and does not require subjective operator intervention/interpretation.
Cell membrane potentials are measured by fast-VSD dyes, upon application of appropriate current stimuli
to the cells, before and after drug administration. Membrane conductance is then extrapolated by local
measurement of the changes in membrane potential.
41
The upper figure shows the instrumentation (A) and an example of the measurement of a single data point.
VSD-stained cells (B) were exposed to increasing concentrations of capsaicin, an agonist of h-TRPV1.
Examples of the analysis of the fluorescence values, before and after capsaicin, are shown as distributions
(C, black and grey traces, respectively) as well as black and white maps of positive and negative variations
(D and D’).
The figure below (A) illustrates an example of capsaicin concentration response curve (-LogEC50 [M]=
8.060; R=0.905). (B) shows that the EC75 concentration of capsaicin is competitively blocked by
capsazepine, an antagonist of h-TRPV1 (-LogIC50 [M]= 7.112; R=0.825).
The platform has been applied to derive experimentally the concentration-response curve of Capsaicin on
the h-TRPV1 receptor and that for GABA on ANS1 differentiated gabaergic neurons. Typically, eight
concentration/response curves (9 point dilution series, each point calculated from several thousand
cells) can be obtained from a 96-well multiwell plate in ~30 min.
Competitive advantages of the approach
Time resolution: changes in membrane conductance (typically caused by drug acting on ion channels) can
be measured in the subsecond time scale, i.e. better than in conventional optical drug screening;
Spatial resolution: it works at the single cell level, thereby allowing the study of conductance changes in
identified cells within heterogeneous populations as well as in subcompartments of cells with complex
morphology;
Sensitivity: it does not require overexpression of channels/transporters to have a proper readout of their
activity;
Specificity: it provides a highly specific readout, with no restrictions on the nature of the molecular
mechanism responsible for the change in conductance;
Scalability: it makes cell-based high-content-screening compatible with the requirements of high
throughput screening.
For further information on this project please contact:
Business Contact
Paola Pozzi
Head, Office of Biotechnology Transfer
San Raffaele Hospital and Scientific
Institute
Tel: +39 02 2643 4987
Fax: + 39 02 2643 5264
Email: [email protected]
Scientific Contacts
Dr. Andrea D. Menegon / Prof. Fabio Grohovaz
Advanced Light and Electron Microscopy BioImaging
Center (Alembic)
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 4637
Fax: + 39 02 2643 4811
Email: [email protected]/[email protected]
42
“Low carbs arginine bar”
Chronic L-arginine oral supplementation has been proven to have beneficial effects over endothelial
function both in healthy individuals and in type 2 diabetic patients especially when it is associated with
physical exercise (Lucotti P et al Am J Physiol Endocrinol Metab 2006). In addition, it was demonstrated
that chronic L-arginine therapy added to a physical exercise and diet program could also improve glucose
metabolism and insulin sensitivity in a population of obese type 2 diabetic patients and in patients with the
Metabolic Syndrome (MS). Furthermore, it improved endothelial function, oxidative status, and adipokine
release (Piatti PM et al. Circulation 2003). Long-term oral L- arginine treatment resulted in an additive
effect compared with a diet and exercise training program alone on glucose metabolism and insulin
sensitivity (Piatti PM et al. Diabetes Care 2001). The use of L-arginine as food supplement to a normal
diet, in relatively large doses, has been proved to have a salutary effect on cardiovascular diseases as
extensively described in literature, both in animal studies and in humans (Maxwell AJ et al.
Cardiovascular Drugs and Therapy 2000; Mendez JD et al. Biomed Pharmacol 2005). L-arginine was
found to be bioavailable and effective in the prevention of impairment of glucose metabolism and
endothelial dysfunction, improving blood flow. Recently, L-arginine was able to ameliorate glucose
metabolism in a 18 months study with a12 month follow-up in subjects with impaired glucose tolerance
and MS (Monti et al, Diabetes Obes Metab. 2012).
Description of Invention and Potential Applications
A new low carbs arginine formulation has been optimized and was successfully tested in chronic studies
(phase 1 clinical trial on diabetic patients at San Raffaele Hospital) showing significant reduction of body
weight (fat mass). An added value of the present food product is the low content of protein, since it was
demonstrated that diets high in protein are associated with an increased diabetes risk, suggesting a
potential role of decreased protein content for diabetes prevention.
The new formulation could be of benefit for subjects with or at risk of cardiovascular disease, endothelial
dysfunction, altered blood pressure, metabolic Syndrome (including patients with insulin resistance
syndrome, hyperinsulinemia, a population at highly increased cardiovascular risk), high level of
triglycerides, low level of HDL cholesterol, obesity, and impaired glucose tolerance or diabetes.
Low carbs arginine bars give sense of fullness and satisfaction and help subjects following a low calorie
diet to lose weight.
The international patent application was published as WO2012052555.
Patent pending in Europe and US.
Clinical study in fifteen obese subjects with
impaired glucose tolerance (IGT) and metabolic
syndrome (MS). Beneficial effects of oral
administration of L-arginine (6.6 g) containing bars
(6 biscuits) for 14 days were evaluated on body
weight composition, amelioration of endothelial
function, insulin activity, i.e. on insulin sensitivity
and insulin secretion, and lipid levels, as compared
to the same food preparation without the addition
of L-arginine. Figure shows changes in body
weight, fat mass and fat free mass as compared to
baseline in obese subjects receiving the food
preparation + 6.6 g of L-arginine (black
histograms) and in subjects receiving food
preparation without L-arginine (white histogram).
To evaluate changes in body weight compositions,
patients' response to food preparation interventions
was calculated as the difference between the
values obtained at the end and at the beginning of
the each food preparation intervention period.
43
Competitive Advantages
The formulation of the invention containing high content of L-Arginine, is characterized by:
-low amount of calories;
-low amount of sugars;
-low content of proteins;
-low temperature process preserves beneficial properties and prevents degradation of L-Arginine;
-gives sense of fullness and satisfaction
-pleasant taste, by the presence of for instance candies orange slices or un-sugared dried blueberries,
raspberry, blackberry.
At difference with commercially available health food bars, the food product of the present invention
comprises very low levels of fructose. In fact, recent evidences in humans also suggest that consuming
fructose may have particularly adverse effects on selective deposition of visceral and ectopic fat, lipid
metabolism, postprandial hypertriglyceridemia, de novo lipogenesis, blood pressure, and insulin
sensitivity, and that this is particularly true in overweight humans.
An added value of the present food product is the low content of protein since it was demonstrated that
diets high in protein are associated with an increased diabetes risk, suggesting a potential role of
decreased protein content for diabetes prevention.
L-Arginine bio-availability is 100% and this food product shows a beneficial effect on endothelial and
vascular function by increasing nitric oxide and its second messenger, cGMP.
An interesting result of the study in obese subjects with IGT and MS was the significant decrease of body
weight in the group receiving the Low Carbs Arginine bar of the present invention, which was quite
completely accounted by a loss of fat mass. The strength of the present study is that the loss of body
weight was achieved without the help of a structured program of physical activity in patients quite
sedentary.
Importantly, in the chronic study performed on 15 obese subjects with impaired glucose tolerance
and metabolic syndrome, the food product added with 6.6 g of L-arginine for 14 days was safe and
was useful in decreasing body weight and fat mass, improving endothelial and vascular function,
ameliorating glucose metabolism, increasing insulin sensitivity, [beta]-cell function and lipid
levels.
Stage of Development
We have a production site certified with the Italian Ministry of Health.
The product can be labeled and marketed with the following European Food Safety Authority (EFSA)
Health Claim: arginine supplement contained in food products improves NO release and endothelial
function. We are currently undertaking preparation of a new EFSA claim application as dietary food for
fat-mass loss.
We seek a potential commercial partner focused on production and distribution of dietary
products for subjects following low calorie diet and subjects with or at risk of obesity, and
cardiovascular disease.
For further information on this project please contact:
Business Contact
Paola Pozzi
Head, Office of Biotechnology Transfer
San Raffaele Hospital and Scientific Institute
Tel: +39 02 2643 4987
Fax: + 39 02 2643 5264
Email: [email protected]
Scientific Contacts
Prof. Emanuele Bosi / Dr. Monti D. Lucilla
Director Department of Internal and Specialistic
Medicine / Cardiodiabetes & core Lab
San Raffaele Hospital and Scientific Institute
Emails:
[email protected];[email protected]
44