Emily hits back at cruel troll in typically sassy style after being branded a 'fat munter' and 'pig' in vile Instagram message Too Hot To Handle season three trailer: 'Wildest ever' batch of scantily-clad, good-looking singles try and fail to not hook-up for prize money Roddy Ricch cancels Saturday Night Live appearance after COVID exposure Christina Aguilera, 41, stuns in a tight snakeskin bodysuit that shows off her svelte figure Will Pete Davidson host The Oscars?
Lindsay Lohan reveals her natural complexion in a make-up free selfie Teresa Giudice's daughter Gia poses with a LIT firecracker in her mouth as she sizzles in a bikini on wild 21st birthday bash in Miami Real Housewives Of Miami newcomer Nicole Martin has become engaged to her boyfriend Anthony Lopez after seven years of dating and one child Reese Witherspoon calls Ina Garten 'the queen of all things' after the Barefoot Contessa affectionately trolled actress for her healthy self-care tips for Christie Brinkley, 67, and her model daughter Sailor, 23, are beach beauties in bikini portraits taken during Turks and Caicos getaway Coachella Harry Styles, Billie Eilish and Kanye West confirmed as headliners Cheeky How Prince Andrew got into this mess..
Mark Wahlberg is launching his own tequila brand: 'We plan on being the best' Roseanne Barr's daughter Jenny Pentland reveals she has PTSD from being 'locked up' in facilities for 'acting out' during troubled teens Rachel Riley cradles daughter Noa, 8 weeks, as she enjoys birthday trip to garden centre with husband Pasha Kovalev and eldest child Maven, 2 Bradley Cooper is New York chic in a black pea coat as he holds hands with his daughter Lea De Seine, four Summer House's Carl Radke confirms romance with co-star and best friend Lindsay Hubbard: 'I'm very happy!
He doesn't care about anyone else' The Young and the Restless star Richard Burgi axed from the soap opera for 'naively and inadvertently' violating COVID policy Brooke Burke, 50, shows off her toned tummy in a tie dye bra top and leggings Fans go wild for Novak's VERY handsome younger siblings Curse of The Vicar of Dibley: Six of the eight main stars have died in the last decade, including Emma Chambers who had a heart attack aged 53 Millie Mackintosh reveals dramatic birth story after norovirus triggered early labour concerns Downing Street staff held a 'lockdown-breaking party' on the eve of Prince Philip's funeral and 'drank into F1 chiefs confirm they have launched a probe into controversial ending of the title-deciding Abu Dhabi Grand British Vogue debuts February issue featuring nine black women who are 'redefining what it means to be a Wearing a facemask makes you more attractive to the opposite sex, study finds Transgender activist Munroe Bergdorf is unveiled as Cosmopolitan UK's 50th anniversary cover star: Model Has 'prancing pony' Rishi Sunak overplayed his hand: Ambitious Chancellor is warned he risks damaging his No10 aides make sure Dilyn still gets his walkies as Boris isolates after family member Will row over BBC's coverage of partygate and Boris Johnson's apology cost the corporation in licence fee Police issue new appeal over murder of Irish primary school teacher, 23, as year-old man arrested for the Finally, product formulation takes place, and the manufactured product is transported back to the clinic to be infused into the patient.
CAR composition: antigen binding domain 1 , transmembrane domain 2 spanning the cell membrane 3 , costimulatory domain 4 , and signaling domain 5. We and others consider virus-free gene transfer technology as a key leverage to define a sustainable market price by facilitating streamlined and cost-effective manufacturing of genetically engineered therapeutic cells.
DNA transposons are genetic elements with the ability to change their positions within the genome [1]. Importantly, it is possible to separate the two functional components of the transposon the TIRs and the transposase in the form of bi-component vector systems reviewed in [2, 3]. Transposon-based vectors enable incorporation of virtually any DNA sequence of interest between the transposon TIRs and mobilization by trans -supplementing the transposase Fig.
In the transposition process, the transposase enzyme mediates the excision of the element from the donor vector, followed by integration of the transposon into a chromosomal locus Fig. This feature uniquely positions transposons as non-viral gene delivery systems capable of efficient genomic integration that can be used as tools for versatile applications in genetic engineering, including gene therapy reviewed in [3].
After binding within the terminal inverted repeats of the transposon TIRs, yellow rectangles flanking a gene of interest GOI, green rectangle , SB transposase blue circles performs the excision of the transposon from the donor DNA black strand and integrates it into a site in the genomic target DNA purple strand. SB was the first transposon ever shown capable of efficient transposition in vertebrate cells, thereby enabling new avenues for genetic engineering, including gene therapy reviewed in [2, 3, 5—14].
SB transposition-based non-viral gene delivery has an outstanding potential to provide innovative and potentially curative treatments for an array of monogenetic disorders reviewed in [5—9, 11—14, 28—33]. To name only some, candidate diseases include inborn errors such as tyrosinemia type I, the mucopolysaccharidoses or lipoprotein receptor deficiency, lung diseases like cystic fibrosis, familial pulmonary fibrosis, primary pulmonary hypertension, clotting disorders like Hemophilia A and B, globinopathies like sickle cell disease, dermatological disorders like various forms of epidermolysis bullosa caused by mutations in certain laminin or collagen genes , muscular dystrophy syndromes, and age-related macular degeneration.
Moreover, cancer is considered a therapeutic target, either through direct targeting of tumor cells or indirectly via adoptive immunotherapy approaches most recently reviewed in [14]. No acute or late toxicities and no exacerbation of graft-versus-host disease GvHD in the allo-setting were observed. CIK cells display an attractive safety profile with minimal occurrence of GvHD after allogeneic cell transplantation; thus, this study provides evidence that donor-derived cells, engineered with the SB transposon, is a safe and valid therapeutic option for ALL patients following allo-HSCT, even if transplanted from mismatched donors.
There are currently a total of 14 active clinical trials in gene therapy making use of SB gene transfer technology with additional ones in the planning [14]. The typical setup for delivery of the SB transposon system into cells is supplying the two components of the vector system as conventional plasmids Fig.
However, the use of naked plasmid DNA is associated with several issues that hamper its implementation for gene therapy. First, the efficiency of plasmid DNA delivery into primary human cells by electroporation or by other DNA transfection technologies is generally low [36, 37].
Indeed, the pilot CAR-T clinical applications based on SB gene transfer relied on the use of plasmid vectors supplying transposon and transposase, thereby limiting the levels of stable gene transfer and consequently mandating a long ex vivo culture to obtain the required dose of CAR-T cells to treat patients [34]. Finally, the presence of an antibiotic resistance gene typically present in plasmid vectors raises safety concerns in the context of gene therapy due to the potential for horizontal gene transfer.
Recent advances in SB vector technology have demonstrated that both the efficiency and safety of SB gene delivery can be addressed by the use of minicircle MC vectors to encode the transposon and either mRNA synthesized in in vitro transcription reactions or recombinant protein to encode the transposase [38—41] Fig. MCs are supercoiled, minimalistic expression cassettes developed for application in non-viral gene delivery. They are derived from their parental plasmids via an intramolecular recombination process, during which the majority of bacterial backbone sequences is depleted from the vector [42].
MC vectors are therefore significantly reduced in size. We consider at least three advantages associated with MC vectors in the context of SB transposon-based gene delivery. First, MCs are superior over plasmids in terms of overall gene transfer efficiency because, due to their smaller size, they cross cellular membranes the cell membrane and the nuclear membrane more efficiently than plasmids [43, 44].
Third, the absence of bacterial plasmid backbone elements in therapeutic vectors is highly relevant in clinical applications, because antibiotic resistance genes included in a therapeutic cell product may raise safety concerns. The use of in vitro transcribed mRNA to encode the transposase offers additional advantages for therapeutic cell engineering.
For example, electroporation of primary human cells, including hematopoietic stem and progenitor cells and T cells, with mRNA was shown to cause significantly reduced cellular toxicity as compared to nucleofection with plasmid DNA [39, 40, 46].
Second, mRNA bypasses the need for transcription and therefore is, upon transfection, immediately available for protein translation in the cytoplasm. Finally, the implementation of an mRNA source for transient delivery of the SB transposase increases the biosafety of this approach, as mRNA does not bear the risk of chromosomal integration, thereby alleviating a potential risk of genomic instability due to prolonged and uncontrollable transposase expression resulting in continuous remobilization of the already integrated SB transposon.
Implementation of the MC technology in conjunction with synthetic mRNA technologies has recently been shown to enable superior stable gene transfer efficiencies in human T cells for advanced CAR-T-cell engineering with SB transposon vectors [39, 47]. The resulting process Fig.
Process development followed the usual sequence of technology transfer of the preclinical process from the research laboratory, identification and qualification of suitable starting materials and raw materials, definition of specifications, and development of the necessary assays including formal assay validation, process upscaling, and process validation.
The gene transfer system consists of two components, as highlighted above, which are co-transfected by electroporation Nucleofector, Lonza, Basel, CH. The CAR cassette Fig. The targeting domain of the CAR is derived from the medicinal anti-SLAMF7-antibody elotuzumab the safety and tolerability of which was shown previously [51]. The cells are processed sequentially, in an A in B clean room environment. On day 14, cells are harvested and the drug product is formulated at a ratio permissible range: 0.
CRL as targets, as well as residual SBX protein by western blot and vector insertion site profile analyses. All of the latter, not specification-defining assays, follow formally established protocols and include relevant controls, but unlike those defining the product specification are not validated according to guidance of the European Pharmacopoeia. There are a number of inclusion and exclusion criteria that patients have to fulfill in order to participate in the CARAMBA clinical trial, e.
Furthermore, patients must have measurable disease markers of myeloma. Because CAR-T-cell therapy can be quite an intensive treatment with potential side effects such as cytokine release syndrome CRS and neurotoxicity, patients are also required to have a good performance status, which is measured using the Eastern Cooperative Oncology Group ECOG score.
Patients must have an ECOG score of less than 2, implying a greater level of fitness, to participate in the trial. Patients must have adequate heart, liver, and kidney function as assessed by an ultrasound and bloodwork prior to determining their eligibility for the CARAMBA trial.
MM is a rare hematologic malignancy of aberrant plasma cells. The conventional treatment of MM comprises chemotherapy and newer agents like proteasome inhibitors; however, over the past decade MM treatment is being redefined by humoral and cellular immunotherapies. Several BCMA CAR-T-cell products are currently in clinical development, and have accomplished very high rates of responses in patients who had relapsed and were unresponsive refractory to all established anti-MM treatments [52].
However, the clinical experience with BCMA CAR-T cells has also exposed several challenges associated with targeting this antigen, and potential mechanisms of relapse or resistance include antigen downregulation or even loss [53].
Several studies have demonstrated high-level, uniform expression of SLAMF7 on malignant plasma cells; a representative image is displayed in Fig. CRL cells. The inset displays a bright field image of the same cells. There are hundreds of entries that emerge on ClinicalTrials. Indeed, clinical trials targeting a wide range of antigens for hematologic and solid tumors are currently under development; thus, it is predicted that the demand for CAR-T-cell therapies will increase in the near future.
However, costs revolving around manufacturing of autologous cell products every single therapeutic cell batch being uniquely suitable only for the patient from whom the cells had been isolated are expected to become a major bottleneck, potentially limiting the access to these novel drugs in entire geographical regions and societies.
In addition, the manufacturing capacities of centralized and highly specialized GMP production facilities will likely be exhausted, especially if CAR-T therapies will become available for more common forms of cancer affecting larger patient populations. Thus, in order to address the significant and foreseeable demand for CAR-T therapies in the near future [58] and to enable broad access to this therapy, manufacturing protocols that are more suitable to reduce production time and costs of the cell product are currently under investigation and, in this regard, the advances made in the CARAMBA project constitute a breakthrough into a new era in CAR-T-cell therapy.
SB holds significant potential to lower the costs associated with manufacturing of gene therapy products such as CAR-T cells, and thus enhancing availability for the general public. SB technology promises several additional advantages over viral gene transfer, namely, lower biosafety level translating to lower infrastructure costs for manufacturing and quality control and high modularity.
Recently, some modifications of the SBX protein have allowed generation of recombinant, soluble SB protein [41], which in the future will allow for even shorter ex vivo cultivation, with the obvious biological and financial benefits.
Given the financially very significant engagement of major pharmaceutical companies in the fields of cell and gene therapy and immuno-oncology with genetically modified typically autologous immune effector cells, the question why academia remains active in this highly competitive field is often posed.
The amount of public research funding available is already quite limited, size and duration of typical grants fall significantly short of what is required for proper pharmaceutical development, and the probability of failure of any such proposal is outsize.
However, for the foreseeable future we anticipate that the majority of innovations in the fields of cell and gene therapy in general and CAR-T-cell therapy in particular will continue to derive from academic institutions until industry develops its own expertise and gets comfortable with the risk and courage that it takes to bring a truly novel cell product from the bench to first clinical application. Ever so often, these ideas will be taken over by industry and developed to marketable medicines, and we predict that some of the innovations in CARAMBA will be among them.
In order to enable risk assessments, component manufacturers granted the Qualified Person of DRK-BSD exceptionally deep insight into their proprietary processes; the generous cooperation of these manufacturers, specifically Plasmid Factory and Lonza, is hereby gratefully acknowledged as it was absolutely integral to the generation of this novel cellular gene therapy product. Read article at publisher's site DOI : Cancers Basel , 13 24 , 07 Dec Cells , 10 12 , 01 Dec Abramson HN.
Immunotargets Ther , , 09 Sep Int J Mol Sci , 22 21 , 08 Nov Cancers Basel , 13 19 , 29 Sep This data has been text mined from the article, or deposited into data resources.
To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation. Blood , 26 , 31 Oct Cited by: 59 articles PMID: Mol Ther , 29 2 , 14 Oct Cited by: 10 articles PMID: To name only some, candidate diseases include inborn errors such as tyrosinemia type I, the mucopolysaccharidoses or lipoprotein receptor deficiency, lung diseases like cystic fibrosis, familial pulmonary fibrosis, primary pulmonary hypertension, clotting disorders like Hemophilia A and B, globinopathies like sickle cell disease, dermatological disorders like various forms of epidermolysis bullosa caused by mutations in certain laminin or collagen genes , muscular dystrophy syndromes, and age-related macular degeneration.
Moreover, cancer is considered a therapeutic target, either through direct targeting of tumor cells or indirectly via adoptive immunotherapy approaches most recently reviewed in [ 14 ]. No acute or late toxicities and no exacerbation of graft-versus-host disease GvHD in the allo-setting were observed.
CIK cells display an attractive safety profile with minimal occurrence of GvHD after allogeneic cell transplantation; thus, this study provides evidence that donor-derived cells, engineered with the SB transposon, is a safe and valid therapeutic option for ALL patients following allo-HSCT, even if transplanted from mismatched donors. There are currently a total of 14 active clinical trials in gene therapy making use of SB gene transfer technology with additional ones in the planning [ 14 ].
The typical setup for delivery of the SB transposon system into cells is supplying the two components of the vector system as conventional plasmids Fig. However, the use of naked plasmid DNA is associated with several issues that hamper its implementation for gene therapy. First, the efficiency of plasmid DNA delivery into primary human cells by electroporation or by other DNA transfection technologies is generally low [ 36 , 37 ].
Indeed, the pilot CAR-T clinical applications based on SB gene transfer relied on the use of plasmid vectors supplying transposon and transposase, thereby limiting the levels of stable gene transfer and consequently mandating a long ex vivo culture to obtain the required dose of CAR-T cells to treat patients [ 34 ].
Finally, the presence of an antibiotic resistance gene typically present in plasmid vectors raises safety concerns in the context of gene therapy due to the potential for horizontal gene transfer. Recent advances in SB vector technology have demonstrated that both the efficiency and safety of SB gene delivery can be addressed by the use of minicircle MC vectors to encode the transposon and either mRNA synthesized in in vitro transcription reactions or recombinant protein to encode the transposase [ 38 , 39 , 40 , 41 ] Fig.
MCs are supercoiled, minimalistic expression cassettes developed for application in non-viral gene delivery. They are derived from their parental plasmids via an intramolecular recombination process, during which the majority of bacterial backbone sequences is depleted from the vector [ 42 ]. MC vectors are therefore significantly reduced in size. We consider at least three advantages associated with MC vectors in the context of SB transposon-based gene delivery.
First, MCs are superior over plasmids in terms of overall gene transfer efficiency because, due to their smaller size, they cross cellular membranes the cell membrane and the nuclear membrane more efficiently than plasmids [ 43 , 44 ].
Third, the absence of bacterial plasmid backbone elements in therapeutic vectors is highly relevant in clinical applications, because antibiotic resistance genes included in a therapeutic cell product may raise safety concerns. The use of in vitro transcribed mRNA to encode the transposase offers additional advantages for therapeutic cell engineering. For example, electroporation of primary human cells, including hematopoietic stem and progenitor cells and T cells, with mRNA was shown to cause significantly reduced cellular toxicity as compared to nucleofection with plasmid DNA [ 39 , 40 , 46 ].
Second, mRNA bypasses the need for transcription and therefore is, upon transfection, immediately available for protein translation in the cytoplasm.
Finally, the implementation of an mRNA source for transient delivery of the SB transposase increases the biosafety of this approach, as mRNA does not bear the risk of chromosomal integration, thereby alleviating a potential risk of genomic instability due to prolonged and uncontrollable transposase expression resulting in continuous remobilization of the already integrated SB transposon. Implementation of the MC technology in conjunction with synthetic mRNA technologies has recently been shown to enable superior stable gene transfer efficiencies in human T cells for advanced CAR-T-cell engineering with SB transposon vectors [ 39 , 47 ].
The resulting process Fig. Process development followed the usual sequence of technology transfer of the preclinical process from the research laboratory, identification and qualification of suitable starting materials and raw materials, definition of specifications, and development of the necessary assays including formal assay validation, process upscaling, and process validation. The gene transfer system consists of two components, as highlighted above, which are co-transfected by electroporation Nucleofector, Lonza, Basel, CH.
The CAR cassette Fig. The targeting domain of the CAR is derived from the medicinal anti-SLAMF7-antibody elotuzumab the safety and tolerability of which was shown previously [ 51 ].
The cells are processed sequentially, in an A in B clean room environment. On day 14, cells are harvested and the drug product is formulated at a ratio permissible range: 0. CRL as targets, as well as residual SBX protein by western blot and vector insertion site profile analyses. All of the latter, not specification-defining assays, follow formally established protocols and include relevant controls, but unlike those defining the product specification are not validated according to guidance of the European Pharmacopoeia.
There are a number of inclusion and exclusion criteria that patients have to fulfill in order to participate in the CARAMBA clinical trial, e. Furthermore, patients must have measurable disease markers of myeloma. Because CAR-T-cell therapy can be quite an intensive treatment with potential side effects such as cytokine release syndrome CRS and neurotoxicity, patients are also required to have a good performance status, which is measured using the Eastern Cooperative Oncology Group ECOG score.
Patients must have an ECOG score of less than 2, implying a greater level of fitness, to participate in the trial. Patients must have adequate heart, liver, and kidney function as assessed by an ultrasound and bloodwork prior to determining their eligibility for the CARAMBA trial.
MM is a rare hematologic malignancy of aberrant plasma cells. The conventional treatment of MM comprises chemotherapy and newer agents like proteasome inhibitors; however, over the past decade MM treatment is being redefined by humoral and cellular immunotherapies. Several BCMA CAR-T-cell products are currently in clinical development, and have accomplished very high rates of responses in patients who had relapsed and were unresponsive refractory to all established anti-MM treatments [ 52 ].
However, the clinical experience with BCMA CAR-T cells has also exposed several challenges associated with targeting this antigen, and potential mechanisms of relapse or resistance include antigen downregulation or even loss [ 53 ].
Several studies have demonstrated high-level, uniform expression of SLAMF7 on malignant plasma cells; a representative image is displayed in Fig. CRL cells. The inset displays a bright field image of the same cells.
There are hundreds of entries that emerge on ClinicalTrials. Indeed, clinical trials targeting a wide range of antigens for hematologic and solid tumors are currently under development; thus, it is predicted that the demand for CAR-T-cell therapies will increase in the near future. However, costs revolving around manufacturing of autologous cell products every single therapeutic cell batch being uniquely suitable only for the patient from whom the cells had been isolated are expected to become a major bottleneck, potentially limiting the access to these novel drugs in entire geographical regions and societies.
In addition, the manufacturing capacities of centralized and highly specialized GMP production facilities will likely be exhausted, especially if CAR-T therapies will become available for more common forms of cancer affecting larger patient populations.
Thus, in order to address the significant and foreseeable demand for CAR-T therapies in the near future [ 58 ] and to enable broad access to this therapy, manufacturing protocols that are more suitable to reduce production time and costs of the cell product are currently under investigation and, in this regard, the advances made in the CARAMBA project constitute a breakthrough into a new era in CAR-T-cell therapy. SB holds significant potential to lower the costs associated with manufacturing of gene therapy products such as CAR-T cells, and thus enhancing availability for the general public.
SB technology promises several additional advantages over viral gene transfer, namely, lower biosafety level translating to lower infrastructure costs for manufacturing and quality control and high modularity. Recently, some modifications of the SBX protein have allowed generation of recombinant, soluble SB protein [ 41 ], which in the future will allow for even shorter ex vivo cultivation, with the obvious biological and financial benefits.
Given the financially very significant engagement of major pharmaceutical companies in the fields of cell and gene therapy and immuno-oncology with genetically modified typically autologous immune effector cells, the question why academia remains active in this highly competitive field is often posed.
The amount of public research funding available is already quite limited, size and duration of typical grants fall significantly short of what is required for proper pharmaceutical development, and the probability of failure of any such proposal is outsize.
However, for the foreseeable future we anticipate that the majority of innovations in the fields of cell and gene therapy in general and CAR-T-cell therapy in particular will continue to derive from academic institutions until industry develops its own expertise and gets comfortable with the risk and courage that it takes to bring a truly novel cell product from the bench to first clinical application.
Ever so often, these ideas will be taken over by industry and developed to marketable medicines, and we predict that some of the innovations in CARAMBA will be among them.
McClintock B. The origin and behavior of mutable loci in maize. Ivics Z, Izsvak Z. The expanding universe of transposon technologies for gene and cell engineering.
Mob DNA. Transposon-mediated genome manipulation in vertebrates. Nat Methods.
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