Background/Objectives: Parkinson’s disease (PD) is a major neurodegenerative disorder with no cure. The goal is to develop an active immunotherapy targeting aggregated alpha synuclein (aSyn), the root cause of PD. TRB-001 is the lead candidate of a novel class of vaccines. It is a peptide/protein conjugate coupled to sugar residues, which is used to target and activate antigen-presenting cells, and addresses aSyn. Methods: A 33-year-old male, diagnosed with PD seven years previously, with a Hoehn & Yahr stage of 1, taking Levodopa/Benserazide (100/25 mg, 6× per day), Rotigotine (8 mg) and Rasagiline (1 mg), amounting to a Levodopa equivalent daily dose (LEDD) of 940 mg, also complicated by impulse control disorder, requested experimental therapy. He received a total of four TRB-001 administrations (weeks 0, 4, 8 and 34) following informed consent. The workup included safety, immunological and clinical parameters. Results: Vaccinations were well tolerated. They induced a high-titer aSyn-specific antibody (Ab) response. Titer increase was associated with a reduction in aSyn plasma levels, suggesting target engagement. The Ab titer and the reduction in aSyn plasma levels were both long-lived. The boost elicited a recall-type Ab titer increase and triggered avidity maturation (factor 7.8). Abs demonstrated a high degree of selectivity for aggregated aSyn (factor 30). Moreover, they were found to preferentially react with tissue from PD brain lysates. The Movement Disorder Society-Sponsored Unified Parkinson’s Disease Rating Scale (MDS-UPDRS) score for the patient remained essentially stable throughout the observation period of 53 weeks. At the time of the boost, the symptomatic PD therapy was simplified to Levodopa/Carbidopa/Entacapone 100/25/200 mg four times a day, amounting to an LEDD of 532 mg. This put an end to the symptoms of the impulse control disorder. Conclusions: Results obtained suggest that this new class of vaccines may yield Ab responses comparable in magnitude and target avidity to the therapeutic setting of monoclonal Abs. TRB-001 is currently being translated to a randomized, placebo-controlled Phase 1B study.

Vaccines are a promising therapy for the treatment of chronic conditions such as pruritus. IL-31 has been identified as an important mediator of itch. By targeting IL-31 signaling with immunotherapy, CP can be effectively alleviated. However, self-antigens such as IL-31 are highly tolerated, which has rendered conventional conjugate vaccines (CCVs) ineffective at generating sufficient antibody (Ab) responses to alleviate CP. Novel Win the Skin Immune System Trick (WISIT) vaccines however have been shown to induce substantially stronger Ab responses than CCVs in Parkinson's Disease, and so may be capable of overcoming IL-31 tolerance to effectively treat CP. In this report, WISIT vaccines presenting ten different IL-31-specific peptides were compared to CCVs presenting the same peptides. Multiple response parameters were assessed, including Ab titers induced, avidity of these Abs, and IL-31 signaling inhibition. Results demonstrated that WISIT vaccines outperform CCVs across all investigated metrics, culminating in the identification of 3 promising candidate WISIT vaccines to be taken forward for further clinical development. This report thus provides evidence that the improved immunogenicity of WISIT vaccines is not disease-specific and that WISIT vaccines may also be translated to treat dermatological disorders. Further preclinical development will be necessary to prepare the identified IL-31 targeting WISIT vaccine candidates for clinical testing.

We aim to repurpose the widely used adjuvant and immunomodulator Alhydrogel TM at a specific dose and schedule (AD04) as a disease-modifying treatment for mild Alzheimer’s disease (AD). AD04 is a potent adjuvant commonly used in vaccines and allergy immunotherapies to augment the magnitude and durability of the immune response. Our preliminary data come from a clinical Phase 2 study in which AD04 served as a control arm. In this study, AD04 showed promising results in delaying hippocampal atrophy and cognitive decline, as well as improving the quality of life in patients with mild AD. A global statistical test (GST) was used to compare the treatment effect of AD04 to other drugs/drug candidates. GST used is a combination of ADAS-cog, ADCS-ADL and the CDR-sb. As such, it combines three important perspectives of three different aspects of AD progression. The effect size seen with AD04 is comparable or better than that of most other available treatments. However, the precise mode of action of subcutaneous (s.c.) administration of AD04 on the brain has not been deciphered. Aged (24 mo) C57BL/6 mice were injected s.c. with AD04 or with PBS, three times, and 3-5 days after the last injection they were tested for fear conditioning. One day later, the mice were sacrificed, and hippocampi and peripheral organs were subjected to proteomics. Young (10wks) mice were used as controls. In the fear conditioning test, AD04-treated old mice, exhibited an improved freezing behavior compared with the PBS-injected mice. Proteomics analysis of the hippocampi revealed that peripheral AD04 administration modulated the production of proteins involved in lipid metabolism regulation and affected microglial phenotype into a phagocytic type. Based on these preclinical results we conclude that AD04 administration induces microglia to phagocytose brain debris, such as apoptotic neurons, synapses and degraded myelin in the aged brain, and in the AD brain also Abeta, extracellular tau tangles and oxidized lipids. By changing the microglial phenotype, AD04, indirectly reduces the neuroinflammation induced by Abeta, tau and neuronal debris, leading to an anti-inflammatory milieu in the brain which protects and rescues the hippocampus from neurodegeneration.

BackgroundThe AFF006 trial (NCT01117818) provided unexpected evidence of benefits of the vaccine adjuvant AD04 (aluminum oxyhydroxide) in patients with early Alzheimer's disease (AD), compared with AD02, a vaccine consisting of a peptide that mimics the N-terminal region of human amyloid-β (Aβ) conjugated with keyhole limpet hemocyanin.ObjectiveThe objective of this post hoc analysis was to assess whether this unexpected benefit of AD04 was an artifact of multiple testing (i.e., type I error inflation) or a robust result.MethodsIn this post hoc assessment, we used permutation testing to estimate type I error inflation due to the evaluation of multiple outcomes in AFF006. Efficacy was assessed using a patient-level global statistical test combining composite endpoints of cognition, function, and global AD. In addition, we examined the observed treatment benefits of AD04 in the context of effects observed in trials of aducanumab, donanemab, and lecanemab, monoclonal anti-Aβ antibodies that received regulatory approval for AD.ResultsThe global statistical test suggested a treatment benefit of AD04 versus ineffective AD02 arms, even after accounting for multiplicity (primary methodology p-value, 0.03; permutation test p-value, 0.02). The observed effect estimates for AD04 compared favorably with approved monoclonal antibodies.ConclusionsPost-hoc analyses are hypothesis generating rather than confirmatory. Adjusting for multiplicity using permutation testing can determine whether post-hoc effects are worth pursuing, or unlikely to be confirmed. These analyses have motivated a follow-up prospective randomized controlled trial, ADVANCE (EudraCT 2022-003532-73), in which optimized AD04 dosing will be compared to placebo in early AD.

Introduction: Levodopa (LD) is the most efficacious antiparkinsonian drug. However, long-term conventional LD treatment of Parkinson’s disease (PD) is frequently associated with motor complications. This can be attributed to pulsatile dopaminergic stimulation given the short LD half-life of conventional dosage forms. Tablets capable of delivering more stable and sustained dopaminergic stimulation would better mimic the brain’s natural dopamine activity. Methods: In this study, 3D screen printing technology was used to manufacture oral dosage forms characterized by the sequential release of Carbidopa and Levodopa. This was achieved by separating the two compounds into different compartments within the same dosage form, which were arranged (LXM.5-1) or formulated (LXM.5-2) in a specific way. Both novel dosage forms were compared to conventional immediate release forms such as Sinemet®. The physicochemical properties of the resulting tablets, LXM.5-1 and LXM.5-2, were assessed in accordance with the USP. Their pharmacokinetic profiles were defined in pigs. Results: The physicochemical properties of LXM.5-1 and LXM.5-2 complied with regulatory requirements. Dissolution studies revealed sequential CD and LD release for both novel dosage forms. They differed regarding the interval between CD and LD release which was shorter for LXM.5-1. PK studies demonstrated that both novel dosage forms exhibited higher LD bioavailability in comparison to Sinemet®, which was 211.36% and 383.64% for LXM.5-1 and LXM.5-2, respectively. Furthermore, blood levels were more stable and sustained, particularly for LXM.5-2. Conclusions: We conclude that 3D screen-printed LXM.5-1 and LXM.5-2 and variations thereof have the potential to transform the pharmacotherapy of Parkinson’s disease.

A threat to human health in developed and, in particular, in developing countries, counterfeit medicines represent the largest identified fraud market worldwide. 3D screen printing (3DSP), an additive manufacturing technology that enables large-scale production, offers unique opportunities to combat counterfeit drugs. One such possibility is the generation of oral dosage forms with a distinct colored inner structure that becomes visible upon breakage and cannot be copied with conventional manufacturing methods. To illustrate this, we designed tablets containing a blue cross. Owing to paste properties and the limited dimensions of the cross, the production process was chosen to be continuous, involving two screen and paste changes. The two pastes (tablet body, cross) were identical except for the blue color of the latter. This ensured the build-up and mechanical stability of the resulting tablets in a mass production environment. The ensuing tablets were found to be uniform in weight and size and to comply with regulatory requirements for hardness, friability, and disintegration time (immediate release). Moreover, all tablets exhibited the covert anticounterfeit feature. The study delivers a proof-of-concept for incorporating complex structures into tablets using 3DSP and showcases the power of the technology offering new avenues for combating counterfeit drugs.

Background Disease modifying therapies (DMTs) may be most beneficial in early disease, when progression is slow and changes small, with clinical relevance difficult to interpret. Objectives Time component tests (TCTs) translate differences between treatments from mean change, vertical distance between longitudinal trajectories, into intuitively understood time saved, horizontal distance between trajectories, which can be readily combined across endpoints in a global TCT (gTCT). Design The value of composites, time savings estimates, and combination scores to optimize measurement and interpretation of DMTs are demonstrated, along with construction details and simulation studies. Setting TCT methods were applied to a randomized phase II clinical trial. Participants Patients with early Alzheimer's disease (N=332). Intervention Three treatment groups with AFFITOPE® AD02 and two control groups with aluminum oxyhydroxide, AD04. Measurements The co-primary efficacy outcomes were an adapted ADAS-Cog (aADAS) and adapted ADCS-ADL (aADL), which were optimized composite scales specific to cognitive and functional domains. A composite based on these two scores was the study's prespecified primary outcome. The CDR-sb and standard non-adapted ADCS-ADL and ADAS-Cog scales were prespecified secondary outcomes. Results The AD04 2 mg group showed some statistically significant effects compared with other study arms. It is unclear whether the observed 3.8-point difference on the composite is clinically meaningful. TCT results show a time savings of 11 months in an 18-month study with AD04 2 mg. Conclusion The relevance of 11 months saved is more universally understood than a mean difference of 3.8 points in the composite outcome. These results suggest that a combination of a composite approach and a time savings interpretation offers a powerful approach for detecting and interpreting disease modifying effects.

3D printing offers new opportunities to customize oral dosage forms of pharmaceuticals for different patient populations, improving patient safety, care, and compliance. Although several notable 3D print technologies have been developed, such as inkjet printing, powder-based printing, selective laser sintering (SLS) printing, and fused deposition modelling (FDM), among others, their capacity is often limited by the number of printing heads. 3D screen-printing (3DSP) is based on a classic flatbed screen printing that is widely used in industrial appli-cations for technical applications. 3DSP can build up thousands of units per screen simultaneously, enabling mass customization of pharmaceuticals. Here, we use 3DSP to investigate two novel paste formulations: immediate-release (IR) and extended-release (ER) using Paracetamol (acetaminophen) as the active pharmaceutical ingre-dient (API). Both disk-shaped and donut-shaped tablets were fabricated using one or both pastes to design drug delivery systems (DDS) with tailored API release profiles. The size and mass of the produced tablets demonstrated high uniformity. Characterization of the tablets physical properties, such as breaking force (25-39 N) and friability (0.002-0.237%), adhering to Ph. Eur (10th edition). Finally, drug release tests with a phosphate buffer at pH 5.8 showed Paracetamol release depended on the IR-and ER paste materials and their respective compartment size of the composite DDS, which can be readily varied using 3DSP. This work further demonstrates the potential of 3DSP to manufacture complex oral dosage forms exhibiting custom release functionalities for mass production.

Abstract Disease modifying therapies (DMTs) are hypothesized to be most beneficial in early disease when progression is slow and mean changes will be small. Therefore, even highly effective therapies will yield small absolute differences whose clinical relevance may be hard to interpret. Time component tests (TCTs) translate differences between treatments in mean change – the vertical distance between longitudinal trajectories, into an intuitively understood metric of time saved – the horizontal distance between trajectories. This corresponds to maintenance of independence with active treatment. DMTs are likely to impact multiple disease domains simultaneously and on the timescale these outcomes can be readily combined in a global time component test (gTCT). Use of gTCTs reflects a critical shift from emphasizing single outcomes and minimally clinically important effects to valuing true disease slowing, and incremental, but permanent benefits on an entire progressive disease. gTCTs are particularly helpful early in clinical development because combining across scales measuring multiple domains reduces noise and improves power. Clinical outcomes, such as ADAS-Cog, ADCS-ADL, and CDR-sb, reflect different aspects of disease progression and convergence of time savings results across these outcomes is evidence of an upstream effect on the cascade of events leading to neurodegeneration. By tailoring the statistical analysis to treatments with disease modifying effects, treatment effect estimates will be more precise thereby increasing statistical power when multiple endpoints are affected. Results will have less power with symptomatic treatments that primarily impact only one endpoint. The TCT was applied to a phase II clinical trial with a composite scale as the primary outcome. The AD04 2 mg group, showed some statistically significant effects compared with other study arms. It is unclear whether the observed 3.8-point difference on the composite measure is clinically meaningful; however, the TCT results show a time savings of 11 months in an 18 month study with AD04 2 mg. The relevance of 11 months saved is more universally understood than a mean difference of 3.8 points in the composite outcome. These results suggest that a combination of a composite approach and a gTCT (time savings) interpretation offers a powerful approach for detecting disease modifying effects.

Loss of elastin due to aging, disease, or injury can lead to impaired tissue function. In this study, de novo tropoelastin (TE) synthesis is investigated in vitro and in vivo using different TE-encoding synthetic mRNA variants after codon optimization and nucleotide modification. Codon optimization shows a strong effect on protein synthesis without affecting cell viability in vitro, whereas nucleotide modifica-tions strongly modulate translation and reduce cell toxicity. Selected TE mRNA variants (3, 10, and 30 & mu;g) are then analyzed in vivo in porcine skin after intradermal application. Administration of 30 & mu;g of native TE mRNA with a me1 111 modification or 10 and 30 & mu;g of unmodified codon-optimized TE mRNA is required to increase TE protein expression in vivo. In contrast, just 3 & mu;g of a codon-optimized TE mRNA variant with the me1 111 modification is able to increase protein expression. Furthermore, skin toxicity is investigated in vitro by injecting 30 & mu;g of mRNA of selected TE mRNA variants into a human full-thickness skin model, and no toxic effects are observed. Thereby, for the first time, an increased dermal TE synthesis by exogenous administration of synthetic mRNA is demonstrated in vivo. Codon optimization of a synthetic mRNA can significantly increase protein expression and therapeutic outcome.

In vitro transcribed messenger ribonucleic acid (mRNA) constitutes an emerging therapeutic class with several clinical applications. This study presents a systematic comparison of different technologies-intradermal injection, microneedle injection, jet injection, and fractional laser ablation-for the topical cutaneous delivery of mRNA. Delivery of Cy5 labeled mRNA and non-labeled enhanced green fluorescent protein (eGFP) expressing mRNA was investigated in a viable ex vivo porcine skin model and monitored for 48 h. Forty 10 µm-thick horizontal sections were prepared from each skin sample and Cy5 labeled mRNA or eGFP expression visualized as a function of depth by confocal laser scanning microscopy and immunohistochemistry. A pixel-based method was used to create a semi-quantitative biodistribution profile. Different spatial distributions of Cy5 labeled mRNA and eGFP expression were observed, depending on the delivery modality; localization of eGFP expression pointed to the cells responsible. Delivery efficiencies and knowledge of delivery sites can facilitate development of efficient, targeted mRNA-based therapeutics.

Additive manufacturing has the potential to revolutionize the pharmaceutical industry, enabling the production of customized drugs for the mass market. However, concerns over scalability, low mechanical resistance, low printing resolution and limited material choices have so far hindered the practical application of current 3D printing (3DP) technologies. Although conceptually similar, each uses its own method to deposit and cure the material. These limitations are highlighted by the fact that there is only a single 3D printed medicine, Spritam® (an antiepileptic) that is currently FDA approved and marketed [1].

Three-dimensional (3D) screen printing was used to fabricate oral dosage forms of different geometry and size. The paste required as starting material for the 3D screen printing process was designed for delayed release and contained the model drug paracetamol (acetaminophen). A prototype screen printing unit was used to fabricate different tablets in a single production process. The resulting tablets were produced with three different sizes and designed geometries (disk, donut, cuboid, oval and grid). Investigation of size and mass of the individual tablets demonstrated high uniformity within the various groups of tablets. Further characterization of their physical properties, such as breaking force and friability, yielded results comparing favorably to conventionally produced tablets. Finally, drug release tests in artificial gastric media showed paracetamol release to depend on the surface-area-to-volume ratio. In conclusion, the study shows the potential of 3D screen printing to fabricate more complex oral dosage forms in the setting of mass production with high reproducibility.

Background: Immunotherapies targeting α-synuclein aim to limit its extracellular spread in the brain and prevent progression of pathology in Parkinson’s disease (PD). PD03A is a specific active immunotherapy (SAIT) involving immunization with a short peptide formulation. Objective: This phase 1 study characterized the safety and tolerability of PD03A in patients with early PD. A key secondary objective was to evaluate immunological activity following immunization. Methods: This was a phase 1 study of two different doses of PD03A versus placebo in PD patients. Patients were randomized (1:1:1) to receive four priming plus one booster vaccination of PD03A 15μg, PD03A 75μg or placebo and were followed for 52 weeks. Results: Overall, 36 patients were randomized, of which 35 received five immunizations and completed the study. All patients experienced at least one adverse event. Transient local injection site reactions affected all but two patients; otherwise most AEs were considered unrelated to study treatment. A substantial IgG antibody response against PD03 was observed with a maximum titer achieved at Week-12. Differences in titers between both active groups versus placebo were statistically significant from the second immunization at Week-8 until Week-52. Conclusion: The safety profile and positive antibody response of PD03A supports the further development of active immunotherapeutic approaches for the treatment of PD.

Self-assembly of solid organs from single cells would greatly expand applicability of regenerative medicine. Stem/progenitor cells can self-organize into micro-sized organ units, termed organoids, partially modelling tissue function and regeneration. Here we demonstrated 3D self-assembly of adult and induced pluripotent stem cell (iPSC)-derived fibroblasts, keratinocytes and endothelial progenitors into both, planar human skin in vivo and a novel type of spheroid-shaped skin organoids in vitro, under the aegis of human platelet lysate. Methods: Primary endothelial colony forming cells (ECFCs), skin fibroblasts (FBs) and keratinocytes (KCs) were isolated from human tissues and polyclonally propagated under 2D xeno-free conditions. Human tissue-derived iPSCs were differentiated into endothelial cells (hiPSC-ECs), fibroblasts (hiPSC-FBs) and keratinocytes (hiPSC-KCs) according to efficiency-optimized protocols. Cell identity and purity were confirmed by flow cytometry and clonogenicity indicated their stem/progenitor potential. Triple cell type floating spheroids formation was promoted by human platelet-derived growth factors containing culture conditions, using nanoparticle cell labelling for monitoring the organization process. Planar human skin regeneration was assessed in full-thickness wounds of immune-deficient mice upon transplantation of hiPSC-derived single cell suspensions. Results: Organoids displayed a distinct architecture with surface-anchored keratinocytes surrounding a stromal core, and specific signaling patterns in response to inflammatory stimuli. FGF-7 mRNA transfection was required to accelerate keratinocyte long-term fitness. Stratified human skin also self-assembled within two weeks after either adult- or iPSC-derived skin cell-suspension liquid-transplantation, healing deep wounds of mice. Transplant vascularization significantly accelerated in the presence of co-transplanted endothelial progenitors. Mechanistically, extracellular vesicles mediated the multifactorial platelet-derived trophic effects. No tumorigenesis occurred upon xenografting. Conclusion: This illustrates the superordinate progenitor self-organization principle and permits novel rapid 3D skin-related pharmaceutical high-content testing opportunities with floating spheroid skin organoids. Multi-cell transplant self-organization facilitates development of iPSC-based organ regeneration strategies using cell suspension transplantation supported by human platelet factors.

Background Robust evidence supports the role of a-synuclein pathology as a driver of neuronal dysfunction in Parkinson's disease. PD01A is a specific active immunotherapy with a short peptide formulation targeted against oligomeric a-synuclein. This phase 1 study assessed the safety and tolerability of the PD01A immunotherapeutic in patients with Parkinson's disease. Methods We did a first-in-human, randomised, phase 1 study of immunisations with PD01A, followed by three consecutive study extensions. Patients aged 45-65 years with a clinical diagnosis of Parkinson's disease (<= years since diagnosis and Hoehn and Yahr Stage 1 to 2), imaging results (dopamine transporter single photon emission CT and MRI) consistent with their Parkinson's disease diagnosis, and on stable doses of Parkinson's disease medications for at least 3 months were recruited at a single private clinic in Vienna, Austria. Patients were randomly assigned (1:1), using a computer-generated sequence with varying block size, to receive four subcutaneous immunisations with either 15 mu g or 75 pg PD01A injected into the upper arms and followed up initially for 52 weeks, followed by a further 39 weeks' follow-up. Patients were then randomly assigned (1:1) again to receive the first booster immunisation at 15 mu g or 75 mu g and were followed up for 24 weeks. All patients received a second booster immunisation of 75 mu g and were followed up for an additional 52 weeks. Patients were masked to dose allocation. Primary (safety) analyses included all treated patients. These four studies were registered with EU Clinical Trials Register, EudraCT numbers 2011-002650-31, 2013-001774-20, 2014-002489-54, and 2015-004854-16. Findings 32 patients were recruited between Feb 14, 2012, and Feb 6, 2013, and 24 were deemed eligible and randomly assigned to receive four PD01A priming immunisations. One patient had a diagnosis change to multiple system atrophy and was withdrawn and two patients withdrew consent during the studies. 21 (87%) of 24 patients received all six immunisations and completed 221-259 weeks in-study (two patients in the 15 mu g dose group and one patient in the 75 mu g dose group discontinued). All patients experienced at least one adverse event, but most of them were considered unrelated to study treatment (except for transient local injection site reactions, which affected all but one patient). Serial MRI assessments also ruled out inflammatory processes. Systemic treatment-related adverse events were fatigue (n=4), headache (n=3), myalgia (n=3), muscle rigidity (n=2), and tremor (n=2). The geometric group mean titre of antibodies against the immunising peptide PD01 increased from 1:46 at baseline to 1:3580 at week 12 in the 15 pg dose group, and from 1:76 to 1:2462 at week 12 in the 75 pg dose group. Antibody titres returned to baseline over 2 years, but could be rapidly reactivated after booster immunisation from week 116 onwards, reaching geometric group mean titres up to 1:20218. Interpretation Repeated administrations of PDO1A were safe and well tolerated over an extended period. Specific active immunotherapy resulted in a substantial humoral immune response with target engagement. Phase 2 studies are needed to further assess the safety and efficacy of PDO1A for the treatment of Parkinson's disease.

Multiple system atrophy (MSA) is a rare and fatal neurodegenerative disease with limited symptomatic treatment options. Aggregation of α-synuclein in oligodendrocytes is believed to be a central mechanism of the neurodegenerative process. PD01A and PD03A are 2 novel therapeutic vaccine candidates containing short peptides as antigenic moieties that are designed to induce a sustained antibody response, specifically targeting pathogenic assemblies of α-synuclein. The objectives of the current study were to evaluate primarily the safety and tolerability of PD01A and PD03A in patients with early MSA. Thirty patients (11 women) were randomized to receive 5 subcutaneous injections of either PD01A (n = 12), PD03A (n = 12), or placebo (n = 6) in this patient- and examiner-blinded, placebo-controlled, 52-week phase 1 clinical trial (ClinicalTrial.gov identifier: NCT02270489). Immunogenicity and clinical scores were assessed as secondary objectives. Twenty-nine patients reported a total of 595 treatment-emergent adverse events (mild or moderate, n = 555; severe, n = 40). Treatment-related adverse events included 190 injection-site reactions typically observed in vaccination trials with similar per-subject incidence in the treatment groups over time. Sustained IgG titers were observed in the PD01A-treated group, and 89% of treated patients developed a PD01-specific antibody response after receiving all injections. Induced antibodies displayed clear reactivity to the α-synuclein target epitope. Titers and antibody responder rate (58%) were lower in the PD03A-treated group. In conclusion, both PD01A and PD03A were safe and well tolerated. PD01A triggered a rapid and long-lasting antibody response that specifically targeted the α-synuclein epitope. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Stem/progenitor cells can self-organize into organoids modelling tissue function and regeneration. Here we demonstrate that human platelet-derived factors can orchestrate 3D self-assembly of clonally expanded adult skin fibroblasts, keratinocytes and endothelial progenitors forming skin organoids within three days. Organoids showed distinct signaling patterns in response to inflammatory stimuli that clearly differed from separated cell types. Human induced pluripotent stem cell (hiPSC)-derived skin cell progenitors also self-assembled into stratified human skin within two weeks, healing deep wounds of immune-deficient mice. Co-transplantation of endothelial progenitors significantly accelerated vascularization. Mechanistically, platelet-derived extracellular vesicles mediated the platelet-derived trophic effects. Long-term fitness of epidermal cells was accelerated further by keratinocyte growth factor mRNA transfection. No tumorigenesis was observed upon xenografting. This permits novel rapid 3D skin-related pharmaceutical testing opportunities and facilitates development of iPSC-based skin regeneration strategies.

INTRODUCTION:Immunotherapeutic approaches targeting amyloid β (Aβ) protein and tau in Alzheimer's disease and α-synuclein (α-syn) in Parkinson's disease are being developed for treating dementia with Lewy bodies. However, it is unknown if single or combined immunotherapies targeting Aβ and/or α-syn may be effective.METHODS:Amyloid precursor protein/α-syn tg mice were immunized with AFFITOPEs® (AFF) peptides specific to Aβ (AD02) or α-syn (PD-AFF1) and the combination.RESULTS:AD02 more effectively reduced Aβ and pTau burden; however, the combination exhibited some additive effects. Both AD02 and PD-AFF1 effectively reduced α-syn, ameliorated degeneration of pyramidal neurons, and reduced neuroinflammation. PD-AFF1 more effectively ameliorated cholinergic and dopaminergic fiber loss; the combined immunization displayed additive effects. AD02 more effectively improved buried pellet test behavior, whereas PD-AFF1 more effectively improved horizontal beam test; the combined immunization displayed additive effects.DISCUSSION:Specific active immunotherapy targeting Aβ and/or α-syn may be of potential interest for the treatment of dementia with Lewy bodies.

Application of in vitro transcribed (IVT) messenger ribonucleic acid (mRNA) is an increasingly popular strategy to transiently produce proteins as therapeutics in a tissue or organ of choice. Here, we focused on the skin and aimed to test if whole human skin tissue explant technology can be used to evaluate the expression efficacy of different IVT Interferon alpha (IFN-α) mRNA constructs in situ, after biolistic delivery. Skin explants were viable and intact for at least five days based on histologic analysis and TUNEL staining. Using GFP reporter mRNA formulations, we found mostly epidermal expression after biolistic delivery. Two out of five sequence-optimized IFN-α mRNA variants resulted in significantly improved IFN-α protein expression in human skin compared to native IFN-α mRNA transfection. IFN-α secretion analysis of the surrounding culture media confirmed these results. We provide a proof-of-concept that IFN-α mRNA delivery into intact human full thickness skin explants can be utilized to test mRNA sequence modifications ex vivo. This approach could be used to develop novel mRNA-based treatments of common epidermal skin conditions including non-melanoma skin cancer, where IFN-α protein therapy has previously shown a strong therapeutic effect.