Search

In this paper, after a systematic presentation of the theoretical background of the European Union regulation, we examine the Hungarian regulation and legal practice (using the websites of five listed Hungarian companies and the Budapest Stock Exchange Corporate Governance Recommendations) in connection with the identification of shareholders by the company, the verifiability of the exercise of shareholder's rights, the remuneration policy and reporting of the public company limited by shares and the approval of legal transactions concluded with related party. We also focus on financial governance and certain viewpoints of product governance, with special attention to product approval process and the issue of greenwashing. We chose to emphasize these issues since one cannot find verbatim, translations of legal acts of European Union on these fields with respect to Hungarian legislation. The other reason is that there are available practical experiences in connection with them, therefore we reflect on different anomalies. Our aim is to highlight the need to transpose and implement a legal instrument with such wide-ranging implications as sustainable finance into a thoughtful and coherent set of rules that permeate the legal system.

Timeshare agreement is a contract that aims to the acquisition of the right to use an accommodation for a period of time on a rotating basis and according to this agreement the consumer, in return for consideration, directly or indirectly acquires the right from the marketer for more than one year to use one or more properties repeatedly for recreation or housing aims for a definite period of time. As for the characteristics of the right of utilization acquired by timeshare agreement, from the aspect of using this right, there is the direct and indirect type: in the first case apart from the title nothing is needed in order to use timeshare, but in the latter case a legal instrument (e.g. membership in an organization) must be applied beside the title in order to live with the right of utilization (the right of utilization is attached to the mentioned legal instrument). The direct right of utilization can be considered as right in rem, contract law and it can be special right of use. The economy prefers the character of right in rem of the timeshare because it is attached to the quality characters (value) of that thing as an article in use. During the regulation of timeshare agreements in Europe the protection of the consumer came into the limelight (see: Scandinavian countries, Benelux states, United Kingdom, Germany, Austria, Hungary, Serbia, Croatia, Bosnia Herzegovina, Montenegro, Romania, Czech Republic, Italy) or the market and tax law aspect became important (see: Greece, Portugal, Spain, France). There is a chaotic situation of timeshare in Europe: similarly to the Hungarian legal situation, in many other European states the private law must face challenges considering the right of use acquired by timeshare agreement, only few countries make a stand for only one legal solution.

In this study, we aimed to determine the efficacy of in vivo chimeric antigen receptor (CAR) T cell therapy, generated by targeted lipid nanoparticles (t-LNPs), as an anti-fibrotic in metabolic dysfunction-associated steatotic liver disease. Hepatic fibrosis is a key predictor of mortality in liver disease, driven by fibrogenic hepatic stellate cells (HSCs). In heart, chimeric antigen receptor (CAR) T cells targeting fibroblast activation protein alpha (FAP) reduce murine cardiac fibrosis. However, the value of this approach in liver is unknown. We explored the anti-fibrotic potential of in vivo-generated anti-FAP CAR T cells in metabolic dysfunction-associated steatohepatitis (MASH), a highly prevalent disease with no approved anti-fibrotic therapies. We first established that FAP expression in both human and murine MASH is specific to HSCs. We then used flow cytometry, Sirius Red morphometry, digital pathology analysis, and single nuclear RNA-sequencing to assess the impact of anti-FAP CAR T cell therapy on murine MASH. Anti-CD5 targeted-LNPs carrying anti-FAPCAR mRNA generate activated, transient anti-FAP CAR T cells, which significantly reduced fibrosis by depleting pro-fibrogenic HSCs, and by modulating immune cells, endothelial cells and hepatocytes in a non-cell autonomous manner to mitigate inflammation and restore hepatic homeostasis. These findings reinforce the potential of in vivo CAR T therapy to attenuate a highly morbid and pervasive liver disease through integrated, multicellular salutary effects., One Sentence Summary: RNA-based treatment transiently reprograms immune cells to target scar-forming cells in fatty liver disease, thus improving liver health overall.

Background: In situ cancer vaccination is a therapeutic approach that involves stimulating the immune system in order to generate a polyclonal, anti-tumor response against an array of tumor neoantigens. Traditionally, in situ vaccination approaches have utilized adenoviral vectors to deliver immune-stimulating genes directly to the tumor microenvironment. Lipid nanoparticle (LNP)-mediated delivery methods offer several advantages over adenoviral delivery approaches, including increased safety, repeated administration potential, and enhanced tumor microenvironment activation. Methods: To explore in situ vaccination using LNPs, we evaluated LNP-mediated delivery of a reporter gene, mCherry, and an immune-stimulating gene, IFNβ, in several in vitro and in vivo models of lung cancer. Results: In vitro experiments demonstrated successful transfection of murine cancer cell lines with LNPs carrying both mCherry and IFN-β mRNA, resulting in high expression levels and IFNβ production. In vivo studies using LLC.ova flank tumors showed that intratumoral injection of IFNβ-mRNA LNPs led to significant IFNβ production within the tumor microenvironment, with minimal systemic exposure. Therapeutic efficacy was evaluated by injecting established LLC.ova flank tumors with IFNβ-mRNA LNPs bi-weekly for two weeks. Treated tumors showed significant growth inhibition compared to controls. Flow cytometric analysis of tumor-infiltrating leukocytes revealed that tumors injected with IFNβ-mRNA LNPs were associated with an increased CD8:CD4 T-cell ratio among lymphocytes, more CD69-expressing CD8 T-cells, and an increased presence of M1 macrophages. Efficacy and an abscopal effect were confirmed in a squamous cell carcinoma model, MOC1. No toxicity was observed. Conclusions: These findings show that intratumoral LNP delivery of immune-stimulating mRNA transcripts, such as IFNβ, can effectively stimulate local anti-tumor immune responses and warrants further investigation as a potential immunotherapeutic approach for cancer.

Abstract: X-linked sideroblastic anemia (XLSA) is a congenital anemia caused by mutations in ALAS2, a gene responsible for heme synthesis. Treatments are limited to pyridoxine supplements and blood transfusions, offering no definitive cure except for allogeneic hematopoietic stem cell transplantation, only accessible to a subset of patients. The absence of a suitable animal model has hindered the development of gene therapy research for this disease. We engineered a conditional Alas2-knockout (KO) mouse model using tamoxifen administration or treatment with lipid nanoparticles carrying Cre-mRNA and conjugated to an anti-CD117 antibody. Alas2-KOBM animals displayed a severe anemic phenotype characterized by ineffective erythropoiesis (IE), leading to low numbers of red blood cells, hemoglobin, and hematocrit. In particular, erythropoiesis in these animals showed expansion of polychromatic erythroid cells, characterized by reduced oxidative phosphorylation, mitochondria's function, and activity of key tricarboxylic acid cycle enzymes. In contrast, glycolysis was increased in the unsuccessful attempt to extend cell survival despite mitochondrial dysfunction. The IE was associated with marked splenomegaly and low hepcidin levels, leading to iron accumulation in the liver, spleen, and bone marrow and the formation of ring sideroblasts. To investigate the potential of a gene therapy approach for XLSA, we developed a lentiviral vector (X-ALAS2-LV) to direct ALAS2 expression in erythroid cells. Infusion of bone marrow (BM) cells with 0.6 to 1.4 copies of the X-ALAS2-LV in Alas2-KOBM mice improved complete blood cell levels, tissue iron accumulation, and survival rates. These findings suggest our vector could be curative in patients with XLSA., (© 2025 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Abstract: α-Thalassemia (AT) is one of the most commonly occurring inherited hematological diseases. However, few treatments are available, and allogeneic bone marrow transplantation is the only available therapeutic option for patients with severe AT. Research into AT has remained limited because of a lack of adult mouse models, with severe AT typically resulting in in utero lethality. By using a lipid nanoparticle (LNP) targeting the receptor CD117 and delivering a Cre messenger RNA (mRNACreLNPCD117), we were able to delete floxed α-globin genes at high efficiency in hematopoietic stem cells (HSC) ex vivo. These cells were then engrafted in the absence or presence of a novel α-globin-expressing lentiviral vector (ALS20αI). Myeloablated mice infused with mRNACreLNPCD117-treated HSC showed a complete knock out (KO) of α-globin genes. They showed a phenotype characterized by the synthesis of hemoglobin H (HbH; also known as β-tetramers or β4), aberrant erythropoiesis, and abnormal organ morphology, culminating in lethality ∼8 weeks after engraftment. Mice infused with mRNACreLNPCD117-treated HSC with at least 1 copy of ALS20αI survived long term with normalization of erythropoiesis, decreased production of HbH, and amelioration of the abnormal organ morphology. Furthermore, we tested ALS20αI in erythroid progenitors derived from α-globin-KO CD34+ cells and cells isolated from patients with both deletional and nondeletional HbH disease, demonstrating improvement in α-globin/β-globin mRNA ratio and reduction in the formation of HbH by high-performance liquid chromatography. Our results demonstrate the broad applicability of LNP for disease modeling, characterization of a novel mouse model of severe AT, and the efficacy of ALS20αI for treating AT., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Effective delivery of mRNA or small molecule drugs to the brain is a significant challenge in developing treatment for acute ischemic stroke (AIS). To address the problem, we have developed targeted nanomedicine to increase drug concentrations in endothelial cells of the blood-brain barrier (BBB) of the injured brain. Inflammation during ischemic stroke causes continuous neuronal death and an increase in the infarct volume. To enable targeted delivery to the inflamed BBB, we conjugated lipid nanocarriers (NCs) with antibodies that bind cell adhesion molecules expressed at the BBB. In the transient middle cerebral artery occlusion mouse model, NCs targeted to vascular cellular adhesion molecule-1 (VCAM) achieved the highest level of brain delivery, nearly two orders of magnitude higher than untargeted ones. VCAM-targeted lipid nanoparticles with luciferase-encoding mRNA and Cre-recombinase showed selective expression in the ischemic brain. Anti-inflammatory drugs administered intravenously after ischemic stroke reduced cerebral infarct volume by 62% (interleukin-10 mRNA) or 35% (dexamethasone) only when they were encapsulated in VCAM-targeted NCs. Thus, VCAM-targeted lipid NCs represent a new platform for strongly concentrating drugs within the compromised BBB of penumbra, thereby ameliorating AIS., Competing Interests: Declaration of interests J.N., P.M.G., V.V.S., H.P., D.W., J.S.B., V.R.M., and O.A.M-C. have pending patent applications fully disclosed by the University of Pennsylvania., (Copyright © 2024. Published by Elsevier Inc.)

Lipid nanoparticles (LNPs) have emerged as the dominant platform for RNA delivery, based on their success in the COVID-19 vaccines and late-stage clinical studies in other indications. However, we and others have shown that LNPs induce severe inflammation, and massively aggravate pre-existing inflammation. Here, using structure-function screening of lipids and analyses of signaling pathways, we elucidate the mechanisms of LNP-associated inflammation and demonstrate solutions. We show that LNPs' hallmark feature, endosomal escape, which is necessary for RNA expression, also directly triggers inflammation by causing endosomal membrane damage. Large, irreparable, endosomal holes are recognized by cytosolic proteins called galectins, which bind to sugars on the inner endosomal membrane and then regulate downstream inflammation. We find that inhibition of galectins abrogates LNP-associated inflammation, both in vitro and in vivo . We show that rapidly biodegradable ionizable lipids can preferentially create endosomal holes that are smaller in size and reparable by the endosomal sorting complex required for transport (ESCRT) pathway. Ionizable lipids producing such ESCRT-recruiting endosomal holes can produce high expression from cargo mRNA with minimal inflammation. Finally, we show that both routes to non-inflammatory LNPs, either galectin inhibition or ESCRT-recruiting ionizable lipids, are compatible with therapeutic mRNAs that ameliorate inflammation in disease models. LNPs without galectin inhibition or biodegradable ionizable lipids lead to severe exacerbation of inflammation in these models. In summary, endosomal escape induces endosomal membrane damage that can lead to inflammation. However, the inflammation can be controlled by inhibiting galectins (large hole detectors) or by using biodegradable lipids, which create smaller holes that are reparable by the ESCRT pathway. These strategies should lead to generally safer LNPs that can be used to treat inflammatory diseases.

The use of lipid nanoparticles (LNP) to encapsulate and deliver mRNA has become an important therapeutic advance. In addition to vaccines, LNP-mRNA can be used in many other applications. For example, targeting the LNP with anti-CD5 antibodies (CD5/tLNP) can allow for efficient delivery of mRNA payloads to T cells to express protein. As the percentage of protein expressing T cells induced by an intravenous injection of CD5/tLNP is relatively low (4-20%), our goal was to find ways to increase mRNA-induced translation efficiency. We showed that T cell activation using an anti-CD3 antibody improved protein expression after CD5/tLNP transfection in vitro but not in vivo. T cell health and activation can be increased with cytokines, therefore, using mCherry mRNA as a reporter, we found that culturing either mouse or human T cells with the cytokine IL7 significantly improved protein expression of delivered mRNA in both CD4 + and CD8 + T cells in vitro. By pre-treating mice with systemic IL7 followed by tLNP administration, we observed significantly increased mCherry protein expression by T cells in vivo. Transcriptomic analysis of mouse T cells treated with IL7 in vitro revealed enhanced genomic pathways associated with protein translation. Improved translational ability was demonstrated by showing increased levels of protein expression after electroporation with mCherry mRNA in T cells cultured in the presence of IL7, but not with IL2 or IL15. These data show that IL7 selectively increases protein translation in T cells, and this property can be used to improve expression of tLNP-delivered mRNA in vivo., Competing Interests: Competing interests statement:B.A.S. and A.B. are employees of Capstan Therapeutics. C.H.J., H.A., D.W., S.M.A., and H.P. are scientific founders and hold equity in Capstan Therapeutics. Authors have patent filings in the field of RNA therapeutics.

RNA therapeutics are an emerging, powerful class of drugs with potential applications in a wide range of disorders. A central challenge in their development is the lack of clear pharmacokinetic (PK)-pharmacodynamic relationship, in part due to the significant delay between the kinetics of RNA delivery and the onset of pharmacologic response. To bridge this gap, we have developed a physiologically based PK/pharmacodynamic model for systemically administered mRNA-containing lipid nanoparticles (LNPs) in mice. This model accounts for the physiologic determinants of mRNA delivery, active targeting in the vasculature, and differential transgene expression based on nanoparticle coating. The model was able to well-characterize the blood and tissue PKs of LNPs, as well as the kinetics of tissue luciferase expression measured by ex vivo activity in organ homogenates and bioluminescence imaging in intact organs. The predictive capabilities of the model were validated using a formulation targeted to intercellular adhesion molecule-1 and the model predicted nanoparticle delivery and luciferase expression within a 2-fold error for all organs. This modeling platform represents an initial strategy that can be expanded upon and utilized to predict the in vivo behavior of RNA-containing LNPs developed for an array of conditions and across species., Competing Interests: H.P. and D.W. are scientific founders and hold equity in Capstan Therapeutics. S.C.S. and B.L.M. are employees and hold equity in Acuitas Therapeutics. H.P. and D.W. receive research support from BioNTech. H.P., V.V.S., D.W., and V.R.M. are named on patents that describe the use of nucleoside-modified mRNA and targeted LNP., (© 2024 The Authors.)

With six therapies approved by the Food and Drug Association, chimeric antigen receptor (CAR) T cells have reshaped cancer immunotherapy. However, these therapies rely on ex vivo viral transduction to induce permanent CAR expression in T cells, which contributes to high production costs and long-term side effects. Thus, this work aims to develop an in vivo CAR T cell engineering platform to streamline production while using mRNA to induce transient, tunable CAR expression. Specifically, an ionizable lipid nanoparticle (LNP) is utilized as these platforms have demonstrated clinical success in nucleic acid delivery. Though LNPs often accumulate in the liver, the LNP platform used here achieves extrahepatic transfection with enhanced delivery to the spleen, and it is further modified via antibody conjugation (Ab-LNPs) to target pan-T cell markers. The in vivo evaluation of these Ab-LNPs confirms that targeting is necessary for potent T cell transfection. When using these Ab-LNPs for the delivery of CAR mRNA, antibody and dose-dependent CAR expression and cytokine release are observed along with B cell depletion of up to 90%. In all, this work conjugates antibodies to LNPs with extrahepatic tropism, evaluates pan-T cell markers, and develops Ab-LNPs capable of generating functional CAR T cells in vivo., (© 2023 Wiley-VCH GmbH.)

Hematopoietic stem cells (HSCs) are the source of all blood cells over an individual's lifetime. Diseased HSCs can be replaced with gene-engineered or healthy HSCs through HSC transplantation (HSCT). However, current protocols carry major side effects and have limited access. We developed CD117/LNP-messenger RNA (mRNA), a lipid nanoparticle (LNP) that encapsulates mRNA and is targeted to the stem cell factor receptor (CD117) on HSCs. Delivery of the anti-human CD117/LNP-based editing system yielded near-complete correction of hematopoietic sickle cells. Furthermore, in vivo delivery of pro-apoptotic PUMA (p53 up-regulated modulator of apoptosis) mRNA with CD117/LNP affected HSC function and permitted nongenotoxic conditioning for HSCT. The ability to target HSCs in vivo offers a nongenotoxic conditioning regimen for HSCT, and this platform could be the basis of in vivo genome editing to cure genetic disorders, which would abrogate the need for HSCT.

After more than 100 failed drug trials for acute ischemic stroke (AIS), one of the most commonly cited reasons for the failure has been that drugs achieve very low concentrations in the at-risk penumbra. To address this problem, here we employ nanotechnology to significantly enhance drug concentration in the penumbra's blood-brain barrier (BBB), whose increased permeability in AIS has long been hypothesized to kill neurons by exposing them to toxic plasma proteins. To devise drug-loaded nanocarriers targeted to the BBB, we conjugated them with antibodies that bind to various cell adhesion molecules on the BBB endothelium. In the transient middle cerebral artery occlusion (tMCAO) mouse model, nanocarriers targeted with VCAM antibodies achieved the highest level of brain delivery, nearly 2 orders of magnitude higher than untargeted ones. VCAM-targeted lipid nanoparticles loaded with either a small molecule drug (dexamethasone) or mRNA (encoding IL-10) reduced cerebral infarct volume by 35% or 73%, respectively, and both significantly lowered mortality rates. In contrast, the drugs delivered without the nanocarriers had no effect on AIS outcomes. Thus, VCAM-targeted lipid nanoparticles represent a new platform for strongly concentrating drugs within the compromised BBB of penumbra, thereby ameliorating AIS., Graphical Abstract: Acute ischemic stroke induces upregulation of VCAM. We specifically targeted upregulated VCAM in the injured region of the brain with drug- or mRNA-loaded targeted nanocarriers. Nanocarriers targeted with VCAM antibodies achieved the highest brain delivery, nearly orders of magnitude higher than untargeted ones. VCAM-targeted nanocarriers loaded with dexamethasone and mRNA encoding IL-10 reduced infarct volume by 35% and 73%, respectively, and improved survival rates.

Current nucleoside-modified RNA lipid nanoparticle (modmRNA-LNP) technology has successfully paved the way for the highest clinical efficacy data from next-generation vaccinations against SARS-CoV-2 during the COVID-19 pandemic. However, such modmRNA-LNP technology has not been characterized in common pre-existing inflammatory or immune-challenged conditions, raising the risk of adverse clinical effects when administering modmRNA-LNPs in such cases. Herein, we induce an acute-inflammation model in mice with lipopolysaccharide (LPS) intratracheally (IT), 1 mg kg -1 , or intravenously (IV), 2 mg kg -1 , and then IV administer modmRNA-LNP, 0.32 mg kg -1 , after 4 h, and screen for inflammatory markers, such as pro-inflammatory cytokines. ModmRNA-LNP at this dose caused no significant elevation of cytokine levels in naive mice. In contrast, shortly after LPS immune stimulation, modmRNA-LNP enhanced inflammatory cytokine responses, Interleukin-6 (IL-6) in serum and Macrophage Inflammatory Protein 2 (MIP-2) in liver significantly. Our report identifies this phenomenon as inflammation exacerbation (IE), which was proven to be specific to the LNP, acting independent of mRNA cargo, and was demonstrated to be time- and dose-dependent. Macrophage depletion as well as TLR3 -/- and TLR4-/- knockout mouse studies revealed macrophages were the immune cells involved or responsible for IE. Finally, we show that pretreatment with anti-inflammatory drugs, such as corticosteroids, can partially alleviate IE response in mice. Our findings characterize the importance of LNP-mediated IE phenomena in gram negative bacterial inflammation, however, the generalizability of modmRNA-LNP in other forms of chronic or acute inflammatory and immune contexts needs to be addressed., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

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., Competing Interests: Declaration of interests H.P., I.T., N.P., V.R.M., and D.W. are inventors on a patent filed on some aspects of this work. Those interests have been fully disclosed to the University of Pennsylvania. All other authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Mutations within over 250 known genes are associated with inherited retinal degeneration. Clinical success following gene-replacement therapy for congenital blindness due to RPE65 mutations establishes a platform for the development of downstream treatments targeting other forms of inherited ocular disease. Unfortunately, several challenges relevant to complex disease pathology and limitations of current gene-transfer technologies impede the development of related strategies for each specific form of inherited retinal degeneration. Here, we describe a gene-augmentation strategy that delays retinal degeneration by stimulating features of anabolic metabolism necessary for survival and structural maintenance of photoreceptors. We targeted two critical points of regulation in the canonical insulin/AKT/mammalian target of rapamycin (mTOR) pathway with AAV-mediated gene augmentation in a mouse model of retinitis pigmentosa. AAV vectors expressing the serine/threonine kinase, AKT3, promote dramatic preservation of photoreceptor numbers, structure, and partial visual function. This protective effect was associated with successful reprogramming of photoreceptor metabolism toward pathways associated with cell growth and survival. Collectively, these findings underscore the importance of AKT activity and downstream pathways associated with anabolic metabolism in photoreceptor survival and maintenance., (Copyright © 2019 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Variants in PRPF31 , which encodes pre-mRNA processing factor 31 homolog, are known to cause autosomal-dominant retinitis pigmentosa (adRP) with incomplete penetrance. However, the majority of mutations cause null alleles, with only two proven pathogenic missense mutations. We identified a novel missense mutation in PRPF31 in a family with adRP., Methods: We performed whole exome sequencing to identify possible pathogenic mutations in the proband of a family with adRP. Available affected family members had a full ophthalmological evaluation including kinetic and two-colour dark adapted static perimetry, electroretinography and multimodal imaging of the retina. Two patients had evaluations covering nearly 20 years. We carried out segregation analysis of the probable mutation, PRPF31 c.590T>C. We evaluated the cellular localisation of the PRPF31 variant (p.Leu197Pro) compared with the wildtype PRPF31 protein., Results: PRPF31 c.590T>C segregated with the disease in this four-generation autosomal dominant pedigree. There was intrafamilial variability in disease severity. Nyctalopia and mid-peripheral scotomas presented from the second to the fourth decade of life. There was severe rod >cone dysfunction. Visual acuity (VA) was relatively intact and was maintained until later in life, although with marked interocular asymmetries. Laboratory studies showed that the mutant PRPF31 protein (p.Leu197Pro) does not localise to the nucleus, unlike the wildtype PRPF31 protein. Instead, mutant protein resulted in punctate localisation to the cytoplasm., Conclusions: c.590T>C is a novel pathogenic variant in PRPF31 causing adRP with incomplete penetrance. Disease may be due to protein misfolding and associated abnormal protein trafficking to the nucleus., Competing Interests: Competing interests: JB is a founder of Gensight Biologics and of Limelight Bio and a scientific (non-equity-holding) founder of Spark Therapeutics. JB holds Sponsored Research Agreements (SRAs) from Biogen, Limelight Bio and REGENXBio. The coauthors report no additional conflicts., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Validation of gene transfer vectors containing tissue-specific promoters in cell-based functional assays poses a formidable challenge for gene therapy product development. Here, we describe a novel approach based on CRISPR/dCas9 transcriptional activation to achieve robust transgene expression from transgene cassettes containing tissue or cell type-specific promoters after infection with AAV vectors in cell-based systems. Guide RNA sequences targeting two promoters that are highly active within mammalian photoreceptors were screened in a novel promoter activation assay. Using this screen, we generated and characterized stable cell lines that co-express dCas9.VPR and top-performing guide RNA candidates. These cells exhibit potent activation of proviral plasmids after transfection or after infection with AAV vectors delivering transgene cassettes carrying photoreceptor-specific promoters. In addition, we interrogated mechanisms to optimize this platform through the addition of multiple guide RNA sequences and co-expression of the universal adeno-associated virus receptor (AAVR). Collectively, this investigation identifies a rapid and broadly applicable strategy to enhance in vitro expression and to evaluate potency of AAV vectors that rely upon cell or tissue-specific regulatory elements.