BackgroundCAR T cell persistence is the only known clinical correlate of activity. Often in solid tumors, CAR T cells fail to persist in patients shortly after infusion. Modulation and maintenance of ...CAR T cell in patients is crucial for this type of therapy to work. We have completed two CAR T cell clinical trials targeting the tumor specific antigen EGFR variant III (EGFRvIII) in glioblastoma (GBM). Clinical trial data indicated that CAR T cell persistent suffered shortly after infusion. One mechanism to maintain and modulate CAR T cell levels is through mRNA vaccination with the CAR T cell antigen.MethodsWe designed a lipid nanoparticle (LNP) carrying the mRNA for the EGFRvIII antigen to boost CAR T cell levels in vivo. We used a combination of in vitro and in vivo assays to test the ability of a truncated EGFRvIII or the corresponding negative control truncated CD19 to activate and boost CAR T cells. Two CARs directed to EGFR were used to evaluate the efficacy of boosting CAR T cell via mRNA LNP vaccination.Results In vitro gene transfer assays were used to determine the ability of mRNA LNPs to transiently transfect target cells. The truncated EGFRvIII and truncated CD19 expressed in transfected cells. In coculture, CAR T cells were able to recognize and be activated by target cells transfected with their cognate antigen. Cytokine production in CAR T cell demonstrated antigen specific activation in vitro. We next evaluated the ability of mRNA LNPs to boost CAR T cells in a murine tumor model. Tumor bearing mice were treated with sub-therapeutic doses of CAR T cells and subsequently boosted twice using mRNA vaccines to EGFRvIII or CD19. Only mice boosted with the EGFRvIII saw a delay in tumor growth and a conferred survival advantage.ConclusionsTaken together, these data demonstrate that mRNA LNP vaccination with CAR T cell antigens can be an effect way to bolster CAR T cells activity in tumor bearing mice. The antigen specific activation of CAR T cell both in vivo and in vitro highlight the utility of this approach in modulating CAR T cell activity. Furthermore, this technology could be used to rescue poor engraftment of CAR T cell in patients.
BackgroundChimeric Antigen Receptor T cells (CARTs) are a powerful anti-cancer therapy, demonstrating success in hematologic malignancies. The development of targeted lipid nanoparticle-mRNA ...(tLNP-mRNA) therapeutics has allowed for the generation of CARTs in situ and may resolve several challenges of conventional ex vivo viral-engineered CAR T cell products including scalability, access and mulitplexing. The in situ T cell transfection rate achieved by tLNP-mRNA varies from 4–20% of T cells expressing the protein of interest. As tLNP-mRNA platform efficacy may critically depend on the number, metabolic state, and localization of engineered T cells, we investigated whether cytokines could enhance protein expression.MethodsWe used tLNPs that target CD5, a marker expressed highly on mouse and human T cells. These tLNPs carried the mRNA for the reporter protein mCherry or encoded a fibroblast activated protein (FAP) targeted CAR. Mouse and human T cells were cultured with IL2, IL7, IL15 or activated using αCD3/CD28. CD5-mCherry-tLNPs or CD5-FAPCAR-tLNPs were added and protein expression was detected using flow cytometry. For in vivo studies, C57BL/6 mice were pretreated with IL7, injected with tLNPs and sacrificed 24 hours later. To analyze the T cell transcriptome, CD8+ T cells were isolated from mouse spleens and cultured with IL2, IL7 or IL15 for 48 hours before being sequenced.ResultsWe found that CD5-mCherry-tLNPs induced protein expression on 10% of resting T cells in vivo and ~15% of T cells in vivo. Culturing mouse and human T cells with IL7 significantly improved CD5-mCherry-tLNPs protein expression in vitro. This also occurred in the in vivo setting as pre-treating mice with IL7 elevated both the proportion and total number of mCherry expressing T cells. FAPCAR expression was also increased by combining CD5-FAPCAR-tLNPs with recombinant IL7. Transcriptomic analysis showed IL7 selectively increased pathways associated with protein translation. The significance of these transcriptomic changes was demonstrated by showing that after electroporation with mRNA, T cells cultured in IL7 produced more protein compared to IL2 or IL15.ConclusionsT cells can be engineered in situ using CD5-targeted tLNPs and IL7 increases the protein expression induced by tLNPs. Our data suggests that the upregulation of translation-associated pathways in T cells by IL7 could be exploited to improve the expression of proteins in situ after tLNP administration. This provides a novel paradigm through which a T cell activating cytokine, instead of lymphodepletion, can potentiate in situ CAR T cell therapy.Ethics ApprovalThis study was approved by The University of Pennsylvania’s Ethics Board; approval number 806099.
The pulmonary vasculature plays an important role in many lung pathologies, such as pulmonary arterial hypertension, primary graft dysfunction of lung transplant, and acute respiratory distress ...syndrome. Therapy for these diseases is quite limited, largely due to dose-limiting side effects of numerous drugs that have been trialed or approved. High doses of drugs targeting the pulmonary vasculature are needed due to the lack of specific affinity of therapeutic compounds to the vasculature. To overcome this problem, the field of targeted drug delivery aims to target drugs to the pulmonary endothelial cells, especially those in pathological regions. The field uses a variety of drug delivery systems (DDSs), ranging from nano-scale drug carriers, such as liposomes, to methods of conjugating drugs to affinity moieites, such as antibodies. These DDSs can deliver small molecule drugs, protein therapeutics, and imaging agents. Here we review targeted drug delivery to the pulmonary endothelium for the treatment of pulmonary diseases. Cautionary notes are made of the risk–benefit ratio and safety—parameters one should keep in mind when developing a translational therapeutic.
Gene therapy as a modern therapeutic approach has not yet advanced to a globally-approved therapeutic approach. Lack of adequate reliable gene delivery system seems to be one of the major reasons ...from the pharmaceutical biotechnology point of view. Main obstacles delaying successful application of human gene therapy are presented in this review. The unique advantages of non-biological gene carriers as compared to their biological counterparts make them ideal alternatives for overcoming extra- and intracellular barriers in a more safely manner. We, therefore, highlight the significant contributions in non-biological gene delivery and favorable characteristics of different design attitudes with focus on in vivo approaches. Bypassing the rapid extracellular enzymatic degradation of genetic materials is covered in extracellular segment of this review with emphasis on PEGylated and targeted formulations. The successful approaches to pave the rest of the way from cellular uptake to intracellular transfer and gene expression of unpacked DNA are also discussed. From these approaches, we emphasize more on optimization of cationic-based polymers and dendrimers, developing newly designed membrane-effective components, and adjusting the hydrophilic-hydrophobic balance of the synthesized vectors
CAR T cells produced in vivo to treat cardiac injury Rurik, Joel G; Tombácz, István; Yadegari, Amir ...
Science (American Association for the Advancement of Science),
2022-Jan-07, 2022-01-07, 20220107, Letnik:
375, Številka:
6576
Journal Article
Recenzirano
Odprti dostop
Fibrosis affects millions of people with cardiac disease. We developed a therapeutic approach to generate transient antifibrotic chimeric antigen receptor (CAR) T cells in vivo by delivering modified ...messenger RNA (mRNA) in T cell–targeted lipid nanoparticles (LNPs). The efficacy of these in vivo–reprogrammed CAR T cells was evaluated by injecting CD5-targeted LNPs into a mouse model of heart failure. Efficient delivery of modified mRNA encoding the CAR to T lymphocytes was observed, which produced transient, effective CAR T cells in vivo. Antifibrotic CAR T cells exhibited trogocytosis and retained the target antigen as they accumulated in the spleen. Treatment with modified mRNA-targeted LNPs reduced fibrosis and restored cardiac function after injury. In vivo generation of CAR T cells may hold promise as a therapeutic platform to treat various diseases.
Recent advances in mRNA technology and its delivery have enabled mRNA-based therapeutics to enter a new era in medicine. The rapid, potent, and transient nature of mRNA-encoded proteins, without the ...need to enter the nucleus or the risk of genomic integration, makes them desirable tools for treatment of a range of diseases, from infectious diseases to cancer and monogenic disorders. The rapid pace and ease of mass-scale manufacturability of mRNA-based therapeutics supported the global response to the COVID-19 pandemic. Nonetheless, challenges remain with regards to mRNA stability, duration of expression, delivery efficiency, and targetability, to broaden the applicability of mRNA therapeutics beyond COVID-19 vaccines. By learning from the rapidly expanding preclinical and clinical studies, we can optimise the mRNA platform to meet the clinical needs of each disease. Here, we will summarise the recent advances in mRNA technology; its use in vaccines, immunotherapeutics, protein replacement therapy, and genomic editing; and its delivery to desired specific cell types and organs for development of a new generation of targeted mRNA-based therapeutics.
Abstract Among synthetic carriers, dendrimers with the more flexible structure have attracted a great deal of researchers’ attention in the field of gene delivery. Followed by the promising results ...upon hydrophobic modification on polymeric structures in our laboratory, alkylcarboxylated poly (propylenimine)-based carriers were synthesized by nucleophilic substitution of amines with alkyl moieties and were further characterized for their physicochemical and biological characteristics for plasmid DNA delivery. Although not noticeably effective gene transfer activity for hexanoate- and hexadecanoate-modified series was observed, but alkylation by decanoic acid significantly improved the transfection efficiency of the final constructs up to 60 fold in comparison with unmodified poly(propylenimine) (PPI). PPI modified by 10-bromodecanoic acid at 50% grafting, showed significantly higher gene expression at c/p ratio of 2 compared to Superfect as positive control. Overall, modification of PPI with 50% primary amines grafting with 10-bromodecanoic acid could increase the transfection efficiency which is occurred at lower c/p ratio when compared to Superfect, i.e. less amount of modified vector is required to exhibit the same efficiency as Superfect. Therefore, the obtained constructs seem to be safer carriers for long-term gene therapy applications.
Ferritin subunits of heavy and light polypeptide chains self-assemble into a spherical nanocage that serves as a natural transport vehicle for metals but can include diverse cargoes. Ferritin ...nanoparticles are characterized by remarkable stability, small and uniform size. Chemical modifications and molecular re-engineering of ferritin yield a versatile platform of nanocarriers capable of delivering a broad range of therapeutic and imaging agents. Targeting moieties conjugated to the ferritin external surface provide multivalent anchoring of biological targets. Here, we highlight some of the current work on ferritin as well as examine potential strategies that could be used to functionalize ferritin via chemical and genetic means to enable its utility in vascular drug delivery.
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Nucleoside-modified messenger RNA (mRNA)-lipid nanoparticles (LNPs) are the basis for the first two EUA (Emergency Use Authorization) COVID-19 vaccines. The use of nucleoside-modified mRNA as a ...pharmacological agent opens immense opportunities for therapeutic, prophylactic and diagnostic molecular interventions. In particular, mRNA-based drugs may specifically modulate immune cells, such as T lymphocytes, for immunotherapy of oncologic, infectious and other conditions. The key challenge, however, is that T cells are notoriously resistant to transfection by exogenous mRNA. Here, we report that conjugating CD4 antibody to LNPs enables specific targeting and mRNA interventions to CD4+ cells, including T cells. After systemic injection in mice, CD4-targeted radiolabeled mRNA-LNPs accumulated in spleen, providing ∼30-fold higher signal of reporter mRNA in T cells isolated from spleen as compared with non-targeted mRNA-LNPs. Intravenous injection of CD4-targeted LNPs loaded with Cre recombinase-encoding mRNA provided specific dose-dependent loxP-mediated genetic recombination, resulting in reporter gene expression in about 60% and 40% of CD4+ T cells in spleen and lymph nodes, respectively. T cell phenotyping showed uniform transfection of T cell subpopulations, with no variability in uptake of CD4-targeted mRNA-LNPs in naive, central memory, and effector cells. The specific and efficient targeting and transfection of mRNA to T cells established in this study provides a platform technology for immunotherapy of devastating conditions and HIV cure.
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Parhiz and colleagues describe a targeting platform to deliver mRNA into CD4+ T cells, a novel way to affect their function. These cells are one of the cornerstones of the immune system and play a role in practically all diseases where immunity is involved (e.g., cancer, allergies, autoimmune diseases, and HIV infection).
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