Cardiac delivery of modified mRNA using lipid nanoparticles: Cellular targets and biodistribution after intramyocardial administration.

Despite research efforts being made towards preserving (or even regenerating) heart tissue after an ischemic event, there is a lack of resources in current clinical treatment modalities for patients with acute myocardial infarction that specifically address cardiac tissue impairment. Modified messenger RNA (modRNA) presents compelling properties that could allow new therapeutic strategies to tackle the underlying molecular pathways that ultimately lead to development of chronic heart failure. However, clinical application of modRNA for the heart is challenged by the lack of effective and safe delivery systems. Lipid nanoparticles (LNPs) represent a well characterized class of RNA delivery systems, which were recently approved for clinical usage in mRNA-based COVID-19 vaccines. In this study, we evaluated the potential of LNPs for cardiac delivery of modRNA. We tested how variations in C12-200 modRNA-LNP composition affect transfection levels and biodistribution after intramyocardial administration in both healthy and myocardial-infarcted mice, and determined the targeted cardiac cell types. Our data revealed that LNP-mediated modRNA delivery outperforms the current state of the art (modRNA in citrate buffer) upon intramyocardial administration in mice, with only minor differences among the formulations tested. Furthermore, we determined both in vitro and in vivo that the cardiac cells targeted by modRNA-LNPs include fibroblasts, endothelial cells and epicardial cells, suggesting that these cell types could represent targets for therapeutic interference with these LNP formulations. These outcomes may serve as a starting point for LNP development specifically for therapeutic mRNA cardiac delivery applications.

Influence of short term storage conditions, concentration methodsand excipients on extracellular vesicle recovery and function.

Extracellular vesicles (EVs) are phospholipid bilayer enclosed vesicles which play an important role in intercellular communication. To date, many studies have focused on therapeutic application of EVs. However, to progress EV applications faster towards the clinic, more information about the physical stability and scalable production of EVs is needed. The goal of this study was to evaluate EV recovery and function after varying several conditions in the isolation process or during storage. Physical stability and recovery rates of EVs were evaluated by measuring EV size, particle and protein yields using nanoparticle tracking analysis, microBCA protein quantification assay and transmission electron microscopy. Western blot analyses of specific EV markers were performed to determine EV yields and purity. EV functionality was tested in an endothelial cell wound healing assay. Higher EV recovery rates were found when using HEPES buffered saline (HBS) as buffer compared to phosphate buffered saline (PBS) during EV isolation. When concentrating EVs, 15 ml spinfilters with a 10 kDa membrane cutoff gave the highest EV recovery. Next, EV storage in polypropylene tubes was shown to be superior compared to glass tubes. The use of protective excipients during EV storage, i.e. bovine serum albumin (BSA) and Tween 20, improved EV preservation without influencing their functionality. Finally, it was shown that both 4 °C and -80 °C are suitable for short term storage of EVs. Together, our results indicate that optimizing buffer compositions, concentrating steps, protective excipients and storage properties may collectively increase EV recovery rates significantly while preserving their functional properties, which accelerates translation of EV-based therapeutics towards clinical application.

Extracellular vesicles for nucleic acid delivery: progress and prospects for safe RNA-based gene therapy.

Nucleic acid-based drugs offer a potentially effective tool for treatment of a variety of diseases, including cancer, cardiovascular diseases, neurological disorders and infectious diseases. However, clinical applications are hindered by instability of RNA molecules in the circulation and lack of efficient vectors that can deliver RNAs to target tissues and into diseased target cells. Synthetic polymer and lipids as well as virus-based vectors are among the most widely explored vehicles for RNA delivery, but clinical progress has been limited as a result of issues related to toxicity, immunogenicity and low efficiency. Most recently, the discovery that extracellular vesicles (EVs) are endogenous RNA carriers, which may display better biocompatibility and higher delivery efficiency as compared with the synthetic systems, has provided a ray of hope in coping with the delivery dilemma, and EV-based gene therapy has already sparked general interest both in academia and industry. In this review, the current knowledge on EV biology and their role in cell-cell communication will be summarized. Promises of EVs as drug carriers and recent technologies on tailoring EVs' biological attributes will be included, and preclinical studies in which EVs have shown promise for therapeutic RNA delivery will be discussed.

PEGylated and targeted extracellular vesicles display enhanced cell specificity and circulation time.

Extracellular vesicles (EVs) are increasingly being recognized as candidate drug delivery systems due to their ability to functionally transfer biological cargo between cells. However, the therapeutic applicability of EVs may be limited due to a lack of cell-targeting specificity and rapid clearance of exogenous EVs from the circulation. In order to improve EV characteristics for drug delivery to tumor cells, we have developed a novel method for decorating EVs with targeting ligands conjugated to polyethylene glycol (PEG). Nanobodies specific for the epidermal growth factor receptor (EGFR) were conjugated to phospholipid (DMPE)-PEG derivatives to prepare nanobody-PEG-micelles. When micelles were mixed with EVs derived from Neuro2A cells or platelets, a temperature-dependent transfer of nanobody-PEG-lipids to the EV membranes was observed, indicative of a 'post-insertion' mechanism. This process did not affect EV morphology, size distribution, or protein composition. After introduction of PEG-conjugated control nanobodies to EVs, cellular binding was compromised due to the shielding properties of PEG. However, specific binding to EGFR-overexpressing tumor cells was dramatically increased when EGFR-specific nanobodies were employed. Moreover, whereas unmodified EVs were rapidly cleared from the circulation within 10min after intravenous injection in mice, EVs modified with nanobody-PEG-lipids were still detectable in plasma for longer than 60min post-injection. In conclusion, we propose post-insertion as a novel technique to confer targeting capacity to isolated EVs, circumventing the requirement to modify EV-secreting cells. Importantly, insertion of ligand-conjugated PEG-derivatized phospholipids in EV membranes equips EVs with improved cell specificity and prolonged circulation times, potentially increasing EV accumulation in targeted tissues and improving cargo delivery.

Intercalating quaternary nicotinamide-based poly(amido amine)s for gene delivery.

In the development of potent polymeric gene carriers for gene therapy, a good interaction between the polymer and the nucleotide is indispensable to form small and stable polyplexes. Polymers with relatively high cationic charge density are frequently used to provide these interactions, but high cationic charge is usually associated with severe cytotoxicity. In this study an alternative, nucleotide specific binding interaction based on intercalation was investigated to improve polymer/pDNA complex formation. For this purpose bioreducible poly(amido amine) copolymers (p(CBA-ABOL/Nic)) were synthesized with different degrees of intercalating quaternary nicotinamide (Nic) groups and amide-substituted derivatives in their side chains. The quaternary nicotinamide group was chosen as intercalating moiety because this group is part of the naturally occurring NAD+ coenzyme and is therefore expected to be non-toxic and non-carcinogenic. The presence of the quaternary nicotinamide moieties in the poly(amido amine) copolymers showed to effectively promote self-assembled polyplex formation already at low polymer/DNA ratios and results in decreased polyplex size and increased stability of the polyplexes. Furthermore, in contrast to the primary amine functionalized analogs the quaternary nicotinamide polymers showed to be non-hemolytic, indicating their compatibility with cell membranes. Polymers with 25% of Nic in the side chains induced GFP expressions of about 4-5 times that of linear PEI, which is comparable with p(CBA-ABOL), the parent PAA without Nic, but at a two- to fourfold lower required polymer dose. N-phenylation of the nicotinamide functionality even further reduces the required polymer dose to form stable polyplexes, which is a major improvement for these kinds of cationic polymers.

Tryptase and histamine metabolites as diagnostic indicators of indolent systemic mastocytosis without skin lesions.

BACKGROUND: Risk indicators of indolent systemic mastocytosis (ISM) in adults with clinical suspicion of ISM without accompanying skin lesions [urticaria pigmentosa (UP)] are lacking. This study aimed at creating a decision tree using clinical characteristics, serum tryptase, and the urinary histamine metabolites methylimidazole acetic acid (MIMA) and methylhistamine (MH) to select patients for bone marrow investigations to diagnose ISM. METHODS: Retrospective data analysis of all adults, in whom bone marrow investigations were performed to diagnose ISM, was carried out. RESULTS: In total, 142 patients were included. SM was absent in all 44 patients with tryptase <10 µg/l, in 45 of 98 (46%) patients with tryptase ≥10 µg/l and in 18 of 52 patients (35%) with tryptase >20 µg/l. Above 43 µg/l, all patients had ISM (n = 11). Male gender, insect venom anaphylaxis as presenting symptom, tryptase, MIMA, and MH were independent ISM predictors. If tryptase was ≥10 µg/l, the diagnostic accuracy of MIMA and MH was high (areas under the ROC curve 0.92). CONCLUSIONS: In suspected patients without UP, the ISM risk is very low (if present at all) if tryptase is <10 µg/l. If tryptase is ≥10 µg/l, this risk depends on MIMA and MH, being low if these are normal, but high if these are elevated. Male gender and insect venom anaphylaxis are additional risk indicators. We recommend refraining from bone marrow examinations in suspected patients without UP if tryptase is <10 µg/l. Our results question the reliability of the minor diagnostic World Health Organization criterion of tryptase >20 µg/l.

Polymeric carrier systems for siRNA delivery.

RNA interference is a technique to induce sequence-specific gene silencing, but is hampered by inefficient delivery of its mediator, short interfering RNA, into target cells. This review describes recent advances in siRNA delivery using polymeric carrier systems. Structural variations that have been applied to these polymers for optimizing their intracellular trafficking are discussed, as well as strategies for stabilization and targeting to diseased tissues in vivo. Recent findings have highlighted safety issues that need to be taken into account in the design of nanoparticles for clinical application.

Targeted delivery of small interfering RNA to angiogenic endothelial cells with liposome-polycation-DNA particles.

Angiogenesis is an attractive target for cancer therapy, due to its central position in tumor growth and development. Vascular Endothelial Growth Factor (VEGF) and its receptors (VEGFRs) play a key role in the angiogenic process. A promising strategy for targeting VEGF-mediated angiogenesis is RNA interference (RNAi) using short interfering RNA (siRNA). However, for efficacious RNAi a well-designed siRNA delivery system is crucial. Liposome-Polycation-DNA (LPD) particles form a promising system for siRNA delivery to tumors. In order to target angiogenic endothelial cells, LPD particles may be modified with a targeting ligand, such as a cyclic Arg-Gly-Asp (RGD) peptide that specifically binds to integrins expressed on tumor-associated endothelial cells. In the current study, RGD-targeted PEGylated LPD particles containing VEGFR-2 siRNA were prepared and optimized with respect to their size and charge by varying protamine content, carrier DNA content for stronger complexation, and PEGylation density. The size of the optimized particles was around 200 nm and the ζ-potential was approximately +20 mV. The uptake and silencing efficacy of the RGD-targeted PEGylated LPD particles were evaluated in H5V cells (murine endothelial cells) and Human Umbilical Vein Endothelial cells (HUVECs). When compared to non-targeted LPD particles, enhanced uptake and silencing of VEGFR-2 expression was observed for RGD-targeted PEGylated LPD particles. In conclusion, the RGD-targeted PEGylated LPD particles containing VEGFR-2 siRNA presented here may be a promising approach for targeting VEGF-mediated angiogenesis in cancer therapy.

Examining the role of Rac1 in tumor angiogenesis and growth: a clinically relevant RNAi-mediated approach.

Angiogenesis, the sprouting of new blood vessels from the pre-existing vasculature, is a well established target in anti-cancer therapy. It is thought that the Rho GTPase Rac1 is required during vascular endothelial growth factor (VEGF)-mediated angiogenesis. In the present study, we have used a clinically relevant RNA interference approach to silence Rac1 expression. Human umbilical vein endothelial cells were transiently transfected with non-specific control siRNA (siNS) or Rac1 siRNA (siRac1) using electroporation or Lipofectamine 2000. Functional assays with transfected endothelial cells were performed to determine the effect of Rac1 knockdown on angiogenesis in vitro. Silencing of Rac1 inhibited VEGF-mediated tube formation, cell migration, invasion and proliferation. In addition, treatment with Rac1 siRNA inhibited angiogenesis in an in vivo Matrigel plug assay. Intratumoral injections of siRac1 almost completely inhibited the growth of grafted Neuro2a tumors and reduced tumor angiogenesis. Together, these data indicate that Rac1 is an important regulator of VEGF-mediated angiogenesis. Knockdown of Rac1 may represent an attractive approach to inhibit tumor angiogenesis and growth.

Optimization of poly(amido amine)s as vectors for siRNA delivery.

By Michael addition polymerization of N,N'-cystaminebisacrylamide (CBA) with variable ratios of 4-amino-1-butanol (ABOL) and ethylene diamine (EDA) or triethylenetetramine (TETA), poly(amido amine) copolymers could be obtained with tunable charge densities. The copolymers were optimized to serve as nonviral vectors in RNA interference (RNAi) to form stable, nanosized polyplexes with siRNA with maximum transfection efficacy. It was observed that at least 20-30% EDA or TETA amino units in the copolymers is necessary to encapsulate siRNA into small and stable polyplexes (< 200 nm). Incorporation of higher amounts of EDA or TETA in the copolymers did not further improve polyplex formation and stability, but the increased cationic charge in these copolymers resulted in increased cytotoxicity and hemolytic activity. Copolymers with 20% EDA showed excellent gene silencing properties in vitro (70% luciferase knockdown in H1299 cells) with negligible cytotoxicity.

[Corticosteroids and the risk of glaucoma]. / Corticosteroïden en het risico van glaucoom.

Corticosteroids are used to treat many diseases and are prescribed by both specialists and general practitioners. One serious side effect of steroid use is glaucoma. This complication, which can cause blindness, is often only discovered at the end stage. Three patients, two women aged 20 and 32 and a man aged 28 developed glaucoma as a result of topical steroid use. It is advisable to examine patients annually if they have a family history of glaucoma and are using steroids regularly in or around the eye. Follow-up should also be considered for patients with a family history of glaucoma who are using systemic steroids regularly, and for all other patients using steroids regularly in or around the eye. All patients on steroids should consult their ophthalmologist speedily if visual symptoms occur.

Primary cutaneous marginal zone B-cell lymphoma: clinical and therapeutic features in 50 cases.

BACKGROUND: Primary cutaneous marginal zone B-cell lymphoma (PCMZL) is a low-grade B-cell lymphoma that originates in the skin, with no evidence of extracutaneous disease. Studies focusing on the optimal treatment of PCMZL have not been published thus far. We describe 50 patients with PCMZL to further characterize clinical characteristics and outcome and, in particular, to evaluate our current therapeutic approach. OBSERVATIONS: The majority of the patients (36/50 [72%]) presented with multifocal skin lesions, and 14 patients (28%) presented with solitary or localized lesions. The initial treatment of patients with solitary lesions consisted of radiotherapy or excision, whereas patients with multifocal lesions received a variety of initial treatments, most commonly radiotherapy and chlorambucil therapy. Cutaneous relapses developed in 19 (48%) of 40 patients who had complete remission and were more common in patients with multifocal disease. After a median period of follow-up of 36 months, 2 patients developed extracutaneous disease, but none of the patients died of lymphoma. CONCLUSIONS: Patients with PCMZL who have solitary lesions can be treated effectively with radiotherapy or excision. For patients with PCMZL who have multifocal lesions, chlorambucil therapy and radiotherapy are suitable therapeutic options. In case of cutaneous relapses, the beneficial effects of treatment should carefully be weighed against the potential adverse effects.

[The rise in fluoroquinolone-resistant Neisseria gonorrhoeae among people attending the Municipal Health Service's clinic for sexually transmitted diseases in Amsterdam, the Netherlands; cefotaxime now first-choice treatment for uncomplicated gonorrhoea]. / Toename van fluorochinolonresistentie bij Neisseria gonorrhoeae onder bezoekers van de soa-polikliniek in Amsterdam; cefotaxim nu als eerste keus aanbevolen voor ongecompliceerde gonorroe.

OBJECTIVE: To report the incidence of gonorrhoea and the development of resistance to Neisseria gonorrhoeae among attendees at the Municipal Health Service's STD-clinic in Amsterdam, The Netherlands, 2000-2003. DESIGN: Descriptive. METHOD: Urethral or cervical swabs for culture for N. gonorrhoea were taken from attendees at the STD-clinic. Depending on reported sexual techniques throat and rectal swabs were also taken. The disk diffusion technique in combination with a beta-lactamase test were used for sensitivity testing. RESULTS. The number of Neisseria gonorrhoeae (NG) isolates collected at the Amsterdam Municipal Health Service's STD-clinic decreased from 1047 in 2002 to 772 in 2003. The number of fluoroquinolone-resistant NG (FRNG) isolates rose from 3 in 2000 to 56 in 2003 (p < 0.001). FRNG isolates amongst men who have sex with men increased from 1 in 568 isolates (0.2%) in 2000 to 50 in 478 isolates (10.5%) in 2003 (p < 0.001). Amongst heterosexual men, FRNG rose from 2 per 275 (0.7%) in 2000 to 16 per 297 (5.4%) in 2002 and dropped to 6 per 190 (3.4%) in 2003 (p = 0.146). No FRNG isolates were found in women in 2003 (2000: 0/180 (0.0%); 2001: 2/160 (1.3%); 2002: 4/183 (2.2%). CONCLUSION: The recent incidence of FRNG among men who have sex with men to over 5% makes ciprofloxacin and other fluoroquinolones obsolete as the first-choice treatment option for uncomplicated gonorrhoea if no antibiogram is available. It is advised to use cefotaxim when an antibiogram is not available (yet).

[Dutch Institute for Health Care Improvement revised guideline, 'Sexually transmitted diseases and neonatal herpes']. / CBO-richtlijn 'seksueel overdraagbare aandoeningen en herpes neonatorum' (herziening).

The Dutch Institute for Health Care Improvement revised guideline, 'Sexually transmitted diseases and neonatal herpes' summarises the current scientific position on the diagnosis and treatment of a great number of sexually transmitted diseases (STD) and neonatal herpes. Symptomatic treatment of suspected Chlamydia trachomatis infection and gonorrhoea without previous diagnosis is not recommended. Treatment can be started immediately, once samples have been taken. Risk groups eligible for screening or proactive testing on C. trachomatis infection include: partners of C. trachomatis-positive persons, visitors of STD clinics, women who will undergo an abortion, mothers of newborns with conjunctivitis or pneumonitis, young persons of Surinam or Antillean descent, young women with new relationships and individuals whose history indicates risky sexual behaviour. A period of 3 months can be adopted between a risky contact and the HIV test (this used to be 6 months), unless post-exposure prophylaxis was used. For the treatment of early syphilis no distinction is drawn between HIV-infected and non-HIV-infected persons. It is no longer recommended that women in labour with a history of genital herpes are tested for the herpes simplex virus. Virological testing of the neonate is only advised if the mother shows signs of genital herpes during delivery.