15 research outputs found
Externally-Controlled Systems for Immunotherapy: From Bench to Bedside
We thank GENYO Institute and LentiStem Biotech for the
support to compile of the necessary information to write this
review. We also thank Fundación Poco Frecuente (FPF) and
Asociación Española de Enfermos con Glucogenosis (AEEG) for
their kindly support.Immunotherapy is a very promising therapeutic approach against cancer that is
particularly effective when combined with gene therapy. Immuno-gene therapy
approaches have led to the approval of four advanced therapy medicinal products
(ATMPs) for the treatment of p53-deficient tumors (Gendicine and Imlygic), refractory
acute lymphoblastic leukemia (Kymriah) and large B-cell lymphomas (Yescarta). In
spite of these remarkable successes, immunotherapy is still associated with severe
side effects for CD19+ malignancies and is inefficient for solid tumors. Controlling
transgene expression through an externally administered inductor is envisioned as a
potent strategy to improve safety and efficacy of immunotherapy. The aim is to develop
smart immunogene therapy-based-ATMPs, which can be controlled by the addition
of innocuous drugs or agents, allowing the clinicians to manage the intensity and
durability of the therapy. In the present manuscript, we will review the different inducible,
versatile and externally controlled gene delivery systems that have been developed and
their applications to the field of immunotherapy. We will highlight the advantages and
disadvantages of each system and their potential applications in clinics.Spanish ISCIII Health Research FundEuropean Union (EU)
PI12/01097
PI15/02015
PI18/00337
PI18/00330CECEyUCSyF of the Junta de Andalucia FEDER/European Cohesion Fund (FSE) for Andalusia
2016000073391-TRA
2016000073332-TRA
PI-57069
PAIDI-Bio326
PI-0014-2016Nicolas Monardes regional Ministry of Health
0006/2018Spanish Government
FPU16/05467
FPU17/02268MCI
DIN2018-01018
Genome-edited adult stem cells: Next-generation advanced therapy medicinal products
Over recent decades, gene therapy, which has enabled the treatment of several incurable
diseases, has undergone a veritable revolution. Cell therapy has also seen major advances
in the treatment of various diseases, particularly through the use of adult stem cells
(ASCs). The combination of gene and cell therapy (GCT) has opened up new opportunities to improve advanced therapy medicinal products for the treatment of several diseases. Despite the considerable potential of GCT, the use of retroviral vectors has major
limitations with regard to oncogene transactivation and the lack of physiological expression. Recently, gene therapists have focused on genome editing (GE) technologies as an
alternative strategy. In this review, we discuss the potential benefits of using GE technologies to improve GCT approaches based on ASCs. We will begin with a brief summary of
different GE platforms and techniques and will then focus on key therapeutic approaches
that have been successfully used to treat diseases in animal models. Finally, we discuss
whether ASC GE could become a real alternative to retroviral vectors in a GCT setting.European Regional Development Fund
(FEDER), Grant/Award Numbers: PI18/01610,
PI18/00330, PI18/00337, grants PI12/01097;
Spanish ISCIII Health Research Fun
Lentiviral vectors for inducible, transactivator-free advanced therapy medicinal products: Application to CAR-T cells
Controlling transgene expression through an externally
administered inductor is envisioned as a potent strategy
to improve safety and efficacy of gene therapy approaches.
Generally, inducible ON systems require a chimeric transcription
factor (transactivator) that becomes activated by
an inductor, which is not optimal for clinical translation
due to their toxicity. We generated previously the first
all-in-one, transactivator-free, doxycycline (Dox)-responsive
(Lent-On-Plus or LOP) lentiviral vectors (LVs) able to control
transgene expression in human stem cells. Here, we
have generated new versions of the LOP LVs and have
analyzed their applicability for the generation of inducible
advanced therapy medicinal products (ATMPs) with special
focus on primary human T cells. We have shown that, contrary
to all other cell types analyzed, an Is2 insulator must
be inserted into the 30 long terminal repeat of the LOP
LVs in order to control transgene expression in human
primary T cells. Importantly, inducible primary T cells
generated by the LOPIs2 LVs are responsive to ultralow
doses of Dox and have no changes in phenotype or function
compared with untransduced T cells. We validated
the LOPIs2 system by generating inducible CAR-T cells
that selectively kill CD19+ cells in the presence of Dox.
In summary, we describe here the first transactivatorfree,
all-one-one system capable of generating Dox-inducible
ATMPs.Spanish ISCIII Health Research FundEuropean Union (EU) PI18/00337
PI21/00298
RD21/0017/0004
PI18/00330
PI17/00672Red TerAvJunta de Andalucia FEDER/European Cohesion Fund (FSE) for AndalusiaSpanish Government PI18/00337
PI21/00298European Union-NextGenerationEU - Maria Zambrano Senior Program RD21/0017/0004
PI18/00330
PI17/00672Ministry of Health 2016000073332-TRA
PI-57069
CARTPI-0001-201
PE-CART-0031-2020
PI-0014-2016
PECART-0027-2020
ProyExcel_00875
PEER-0286-2019European Cooperation in Science and Technology (COST) 00123009/SNEO-20191072MINECO - European Regional Development Fund PLEC2021-008094Spanish Government 0006/2018FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades CA21113Spanish Government SAF2015-71589-PMCI RYC-2016-21395German Research Foundation (DFG) PY20_00619 y A-CTS-28_UGR20Biomedicine Program of the University of Granada (Spain) FPU16/05467
FPU17/02268
FPU17/04327
DIN2018-010180
DIN2020-011550
PEJ-2018-001760-
Efficacy and safety of universal (TCRKO) ARI-0001 CAR-T cells for the treatment of B-cell lymphoma
Autologous T cells expressing the Chimeric Antigen Receptor (CAR) have been
approved as advanced therapy medicinal products (ATMPs) against several
hematological malignancies. However, the generation of patient-specific CART
products delays treatment and precludes standardization. Allogeneic off-theshelf
CAR-T cells are an alternative to simplify this complex and timeconsuming
process. Here we investigated safety and efficacy of knocking out
the TCR molecule in ARI-0001 CAR-T cells, a second generation aCD19 CAR
approved by the Spanish Agency of Medicines and Medical Devices (AEMPS)
under the Hospital Exemption for treatment of patients older than 25 years with
Relapsed/Refractory acute B cell lymphoblastic leukemia (B-ALL). We first
analyzed the efficacy and safety issues that arise during disruption of the TCR
gene using CRISPR/Cas9. We have shown that edition of TRAC locus in T cells
using CRISPR as ribonuleorproteins allows a highly efficient TCR disruption
(over 80%) without significant alterations on T cells phenotype and with an
increased percentage of energetic mitochondria. However, we also found that
efficient TCRKO can lead to on-target large and medium size deletions,
indicating a potential safety risk of this procedure that needs monitoring.
Importantly, TCR edition of ARI-0001 efficiently prevented allogeneic
responses and did not detectably alter their phenotype, while maintaining a
similar anti-tumor activity ex vivo and in vivo compared to unedited ARI-0001 CAR-T cells. In summary, we showed here that, although there are still some
risks of genotoxicity due to genome editing, disruption of the TCR is a feasible
strategy for the generation of functional allogeneic ARI-0001 CAR-T cells. We
propose to further validate this protocol for the treatment of patients that do
not fit the requirements for standard autologous CAR-T cells administration.Spanish ISCIII Health Research FundEuropean Commission PI15/02015
PI18/00337
PI21/00298Red TerAv RD21/ 0017/0004
PI18/ 00330
PI17/00672CECEyU and CSyF of the Junta de Andalucia FEDER/European Cohesion Fund (FSE) for Andalusia 2016000073391-TRA
2016000073332-TRA
PI-57069
PAIDIBio326
CARTPI-0001- 201
PECART-0031-2020
PI0014-2016
PEER-0286-2019Spanish Government 00123009/SNEO-20191072
PLEC2021-008094regional Ministry of Health 0006/2018
C2-0002-2019Spanish Government FPU16/05467
FPU17/02268
FPU17/04327Junta de Andalucia PECART-00312020Consejeria de Salud y Familias PECART-0027-2020
MCI DIN2018-010180
DIN2020-01155
Physiological lentiviral vectors for the generation of improved CAR-T cells
Anti-CD19 chimeric antigen receptor (CAR)-T cells have
achieved impressive outcomes for the treatment of relapsed
and refractory B-lineage neoplasms.However, important limitations
still remain due to severe adverse events (i.e., cytokine
release syndrome and neuroinflammation) and relapse of
40%–50%of the treated patients.MostCAR-Tcells are generated
using retroviral vectors with strong promoters that lead to high
CAR expression levels, tonic signaling, premature exhaustion,
and overstimulation, reducing efficacy and increasing side effects.
Here, we show that lentiviral vectors (LVs) expressing the
transgene through a WAS gene promoter (AW-LVs) closely
mimic the T cell receptor (TCR)/CD3 expression kinetic upon
stimulation. These AW-LVs can generate improved CAR-T cells
as a consequence of theirmoderate andTCR-like expression profile.
Compared with CAR-T cells generated with human elongation
factor a (EF1a)-driven-LVs, AW-CAR-T cells exhibited
lower tonic signaling, higher proportion of naive and stem cell
memory T cells, less exhausted phenotype, and milder secretion
of tumor necrosis factor alpha (TNF-a) and interferon (IFN)-ɣ
after efficient destruction of CD19+ lymphoma cells, both
in vitro and in vivo.Moreover, we also showed their improved efficiency
using an in vitro CD19+ pancreatic tumor model. We
finally demonstrated the feasibility of large-scale manufacturing
ofAW-CAR-T cells in good manufacturing practice (GMP)-like
conditions. Based on these data, we propose the use of AW-LVs
for the generation of improved CAR-T products.Spanish ISCIII Health Research FundEuropean Commission PI15/02015
PI18/00337
PI21/00298
RD21/0017/0004
PI18/00330
PI17/00672CSyF of the Junta de Andalucia FEDER/European Cohesion Fund (FSE) for Andalusia 2016000073391-TRA
2016000073332-TRA
PI-57069
PA IDI-Bio326
CARTPI-0001-201
PECART-0031-2020
Red RANTECAR CAR-T 2019 00400200101918
PLEC2021-008094
PI-0014-2016
PEER-0286-2019Spanish Government PLEC2021-008094
00123009/SNEO-20191072Nicolas Monardes contracts from regional Ministry of Health 0006/2018
C2-0002-2019German Research Foundation (DFG) FPU16/05467
FPU17/02268
FPU17/04327
MCI DIN2018-010180Fundacion Andaluza Progreso y SaludGerman Research Foundation (DFG) PEJ-2018-001760-AJunta de Andalucia PE-0223-2018Biomedicine Programme of the University of Granada (Spain
Optimización de la inmunoterapia antitumoral con células CAR-T mediante el control de la expresión génica
Con la intención de contribuir en la mejora de las estrategias de inmunoterapia, el objetivo
principal de la presente tesis doctoral persigue mejorar la funcionalidad de las células CAR-T
mediante la optimización de las herramientas de modificación genética que se utilizan para su
generación. Así, nos planteamos la optimización y desarrollo de vectores lentivirales orientados
a incrementar la seguridad y eficacia de las células CAR-T mediante el control de la expresión
génica a través de dos abordajes diferentes, ya sea de forma endógena (regulado por el estado de
activación de la propia célula T) o exógena (mediante la administración de doxiciclina).
Los resultados obtenidos con el sistema LOP-Is2 determinan su potencial y versatilidad
como herramienta para regular la expresión de diferentes transgenes en células T primarias
humanas en un entorno clínico debido a la ausencia de transactivadores y a las bajas dosis de
doxiciclina requeridas.
Esto abre la puerta a explorar nuevas estrategias de inmunoterapia para poder expresar de
forma controlada, nuevas moléculas proteicas que, por su toxicidad, no han podido considerarse
para ser expresados por las células CAR-T o en otros tipos celulares como los linfocitos
infiltrantes del tumor (TILs).Tesis Univ. Granada
Using Gene Editing Approaches to Fine-Tune the Immune System
Genome editing technologies not only provide unprecedented opportunities to study
basic cellular system functionality but also improve the outcomes of several clinical
applications. In this review, we analyze various gene editing techniques used to finetune
immune systems from a basic research and clinical perspective. We discuss
recent advances in the development of programmable nucleases, such as zinc-finger
nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered
regularly interspaced short palindromic repeat (CRISPR)-Cas-associated nucleases. We
also discuss the use of programmable nucleases and their derivative reagents such
as base editing tools to engineer immune cells via gene disruption, insertion, and
rewriting of T cells and other immune components, such natural killers (NKs) and
hematopoietic stem and progenitor cells (HSPCs). In addition, with regard to chimeric
antigen receptors (CARs), we describe how different gene editing tools enable healthy
donor cells to be used in CAR T therapy instead of autologous cells without risking
graft-versus-host disease or rejection, leading to reduced adoptive cell therapy costs
and instant treatment availability for patients. We pay particular attention to the delivery
of therapeutic transgenes, such as CARs, to endogenous loci which prevents collateral
damage and increases therapeutic effectiveness. Finally, we review creative innovations,
including immune system repurposing, that facilitate safe and efficient genome surgery
within the framework of clinical cancer immunotherapies.Spanish ISCIII Health Research FundEuropean Union (EU)
PI12/01097
PI15/02015
PI18/00337
PI18/00330CECEyU and CSyF councils of the Junta de Andalucia FEDER/European Cohesion Fund (FSE)
2016000073391-TRA
2016000073332-TRA
PI-57069
PAIDI-Bio326
PI-0014-2016Nicolas Monardes regional Ministry of Health
0006/2018Spanish Government
FPU16/05467
FPU17/02268Industrial Doctorate Plan MCI
DIN2018-010180SMSI
PEJ-2018-001760-ALentiStem Biotec
Characterization of human peritoneal monocyte/macrophage subsets in homeostasis: Phenotype, GATA6, phagocytic/oxidative activities and cytokines expression
Abstract Peritoneal macrophages play a critical role in the control of infectious and inflammatory diseases. Although recent progress on murine peritoneal macrophages has revealed multiple aspects on their origin and mechanisms involved in their maintenance in this compartment, little is known on the characteristics of human peritoneal macrophages in homeostasis. Here, we have studied by flow cytometry several features of human peritoneal macrophages obtained from the peritoneal cavity of healthy women. Three peritoneal monocyte/macrophage subsets were established on the basis of CD14/CD16 expression (CD14++CD16−, CD14++CD16+ and CD14highCD16high), and analysis of CD11b, CD11c, CD40, CD62L, CD64, CD80, CD86, CD116, CD119, CD206, HLA-DR and Slan was carried out in each subpopulation. Intracellular expression of GATA6 and cytokines (pro-inflammatory IL-6 and TNF-α, anti-inflammatory IL-10) as well as their phagocytic/oxidative activities were also analyzed, in an attempt to identify genuine resident peritoneal macrophages. Results showed that human peritoneal macrophages are heterogeneous regarding their phenotype, cell complexity and functional abilities. A direct relationship of CD14/CD16 expression, intracellular content of GATA6, and activation/maturation markers like CD206 and HLA-DR, support that the CD14highCD16high subset represents the mature phenotype of steady-state human resident peritoneal macrophages. Furthermore, increased expression of CD14/CD16 is also related to the phagocytic activity
Exosome: A New Player in Translational Nanomedicine
Exosomes are extracellular vesicles released by the vast majority of cell types both in vivo and ex vivo, upon the fusion of multivesicular bodies (MVBs) with the cellular plasma membrane. Two main functions have been attributed to exosomes: their capacity to transport proteins, lipids and nucleic acids between cells and organs, as well as their potential to act as natural intercellular communicators in normal biological processes and in pathologies. From a clinical perspective, the majority of applications use exosomes as biomarkers of disease. A new approach uses exosomes as biologically active carriers to provide a platform for the enhanced delivery of cargo in vivo. One of the major limitations in developing exosome-based therapies is the difficulty of producing sufficient amounts of safe and efficient exosomes. The identification of potential proteins involved in exosome biogenesis is expected to directly cause a deliberate increase in exosome production. In this review, we summarize the current state of knowledge regarding exosomes, with particular emphasis on their structural features, biosynthesis pathways, production techniques and potential clinical applications.This research was funded by the Spanish ISCIII Health Research Fund and the European Regional Development Fund (FEDER) through research grants PI12/01097, PI15/02015, PI18/00337 (F.M.), PIE16-00045 (J.A.M.), DTS19/00145 (J.A.M.) and PI18/00330 (K.B.), as well as by the Spanish Ministry of Science, Innovation and Universities (MICIU) through FEDER research grant RTI2018-101309-B-C2 (J.A.M.). The CECEyU and CSyF Councils of the Junta de Andalucía FEDER/European Cohesion Fund (FSE) provided the following research grants: 2016000073391-TRA, 2016000073332-TRA, PI-57069 and PAIDI-Bio326 (F.M.) and PI-0014-2016 (K.B). K.B. was also on a Nicolas Monardes Regional Ministry of Health contract (0006/2018). H.A. is supported by Research Excellence PhD Fellowship (2UAE2020) from the National Center for Scientific and Technical Research (CNRST). M.T.-M. is funded by the Spanish Ministry of Science and Innovation (SMSI) through an FPU16/05467 fellowship. M.C.-G. is funded by SMSI through a GJ fellowship (PEJ-2018-001760-A).Ye
Improved Functionality of Integration-Deficient Lentiviral Vectors (IDLVs) by the Inclusion of IS2 Protein Docks
This study was funded by the Spanish ISCIII Health Research Fund and the European Regional Development Fund (FEDER) through research grants PI12/01097, PI15/02015, PI18/00337 (F.M.) PIE16-00045 (J.A.M.), DTS19/00145 (J.A.M.), and PI18/00330 (K.B.) The CECEyU and CSyF of the Junta de Andalucia FEDER/European Cohesion Fund (FSE) for Andalusia provided the following research grants: 2016000073391-TRA, 2016000073332-TRA, PI-57069, and PAIDI-Bio326 (F.M.) and PI0014-2016 (K.B.). K.B. also held a Nicolas Monardes regional Ministry of Health contract (0006/2018). M.T.-M. and N.M.-P. are funded by the Spanish Ministry of Science and Innovation (SMSI) through fellowships FPU16/05467 and FPU17/02268, respectively. M.C.-G is funded by the SMSI through fellowship (PEJ-2018-001760-A). The Ramon y Cajal grant RYC-2015-18382 to P.J.R. founded by the Ministry of Economy and Competitiveness. L.L.-O. is supported by the University of Granada doctoral program (2017). M.C.-G., M.T.-M. and N.M.-P. are University of Granada Biomedicine PhD students.Integration-deficient lentiviral vectors (IDLVs) have recently generated increasing interest,
not only as a tool for transient gene delivery, but also as a technique for detecting off-target cleavage
in gene-editing methodologies which rely on customized endonucleases (ENs). Despite their broad
potential applications, the efficacy of IDLVs has historically been limited by low transgene expression
and by the reduced sensitivity to detect low-frequency off -target events. We have previously reported
that the incorporation of the chimeric sequence element IS2 into the long terminal repeat (LTR) of
IDLVs increases gene expression levels, while also reducing the episome yield inside transduced
cells. Our study demonstrates that the effectiveness of IDLVs relies on the balance between two
parameters which can be modulated by the inclusion of IS2 sequences. In the present study, we
explore new IDLV configurations harboring several elements based on IS2 modifications engineered
to mediate more efficient transgene expression without affecting the targeted cell load. Of all the
insulators and configurations analysed, the insertion of the IS2 into the 30LTR produced the best
results. After demonstrating a DAPI-low nuclear gene repositioning of IS2-containing episomes,
we determined whether, in addition to a positive effect on transcription, the IS2 could improve the
capture of IDLVs on double strand breaks (DSBs). Thus, DSBs were randomly generated, using the
etoposide or locus-specific CRISPR-Cas9. Our results show that the IS2 element improved the efficacy
of IDLV DSB detection. Altogether, our data indicate that the insertion of IS2 into the LTR of IDLVs
improved, not only their transgene expression levels, but also their ability to be inserted into existing
DSBs. This could have significant implications for the development of an unbiased detection tool for
off-target cleavage sites from different specific nucleases.Spanish ISCIII Health Research FundEuropean Commission PI12/01097
PI15/02015
PI18/00337
PIE16-00045
DTS19/00145
PI18/00330Junta de Andalucia 2016000073391-TRA
2016000073332-TRA
PI-57069
PAIDI-Bio326
PI0014-2016Nicolas Monardes regional Ministry of Health 0006/2018Spanish Government FPU16/05467SMSI through fellowship PEJ-2018-001760-ASpanish Government RYC-2015-18382Ministry of Economy and CompetitivenessUniversity of Granada doctoral progra