Background: Scalability and cost remain major manufacturing barriers limiting broad patient access to adeno-associated virus (AAV) gene therapies. While capsid engineering has advanced vector biology, comparatively fewer innovations have addressed fundamental upstream productivity constraints. Transient triple-plasmid transfection is still the dominant AAV production platform and relies on large bacterial backbone plasmids that impose DNA burden and contribute significantly to the cost-of-goods. Methods: In this study, we evaluated a compact NanoplasmidTM DNA system (Aldevron) as a structural redesign of the transfection substrate to enhance upstream productivity. Conventional pUC-based triple-plasmid systems were compared to fully substituted NanoplasmidTM equivalents across suspension HEK293 production platforms optimised via response surface Design of Experiments. Hybrid plasmid configurations were also constructed to assess component-level contributions. Results: Complete substitution with NanoplasmidTM resulted in up to a 10-fold increase in vector genome titre relative to conventional plasmids under matched conditions. Hybrid systems failed to recapitulate this improvement, demonstrating that full-system backbone minimisation is required to ensure high yield. Productivity gains were preserved across transfection reagents and suspension media. NanoplasmidTM deployment represents a scalable, capsid-independent upstream intensification approach that improves yield without altering capsid biology. Conclusions: Integration of this approach within a design-for-manufacturability framework offers a practical route to reducing bacterial plasmid elements increasing safety, enhancing process robustness, and improving economic feasibility of AAV therapeutics.

CLN5 disease, caused by mutations in the CLN5 gene, is a form of neuronal ceroid lipofuscinoses (Batten disease). Patients suffer progressive motor dysfunction, vision loss, seizures, and dementia, leading to premature death. Here, we report a preclinical study of AAV9-mediated gene therapy in a Cln5-/- mouse model. Single-dose AAV9 carrying human CLN5 driven by the CAG or human synapsin 1 promoter (hSYN) was administered via intracerebroventricular injection into neonatal and juvenile Cln5-/- mice. Treatment efficacy was evaluated by assessment of neurodegeneration, neuroinflammation, locomotor function, and survival. AAV9 expressing CLN5 driven by the hSYN promoter significantly alleviated neurodegeneration, improved biochemical and glycosphingolipid profiles, neuropathological and locomotor function, and extended lifespan of the Cln5-/- mice. However, gene transfer employing the CAG promoter demonstrated limited therapeutic efficacy. Furthermore, delayed intervention in juveniles provided superior therapeutic response compared with early neonatal intervention and normalized lifespan. Finally, blood plasma neurofilament light that is significantly elevated in the Cln5-/- mice is restored to normal wildtype levels following treatment. These results indicate that brain-directed adeno-associated virus (AAV) gene therapy could be a promising treatment strategy for CLN5 disease and efficacy might be monitored using a noninvasive blood plasma biomarker.

Inherited biallelic mutations in the CLN7 gene result in the variant late infantile onset neuronal ceroid lipofuscinosis, a subtype of Batten disease (BD), a severe and fatal childhood neurodegenerative disease. Intriguingly, CLN7 genetic variants have also been associated with retinopathies, amyotrophic lateral sclerosis, and frontotemporal dementia. CLN7 encodes a transmembrane protein localizing to endolysosomal membranes with outward-facing chloride channel activity. Loss of CLN7 function results in cortical neurons accumulating swollen lipofuscin-containing lysosomes, leading to neuroinflammation and neurodegeneration. The molecular mechanisms underlying CLN7 BD neuropathology are not completely understood. We have generated iPSC lines from two CLN7 BD patients and age-matched unaffected controls to interrogate intracellular molecular phenotypes in iPSC-derived neural progenitor cells (iNPC). Taking a multi-omics approach we have identified disease-modified activities in endolysosomal transport in iNPCBD that lead to lysosomal dysfunction and decreased mitophagy, resulting in the accumulation of metabolically defective mitochondria. We further observe a breakdown in nuclear functions that centre on RNA processing and nuclear export, linking to CLN7 protein interactions at the stress granule. We have identified dual and distinct functions for CLN7, promoting cell survival during the cellular stress response. CLN7 loss of function in BD results in neuronal apoptosis.

The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is an intronic G4C2 repeat expansion in C9orf72. The repeats undergo bidirectional transcription to produce sense and antisense repeat RNA species, which are translated into dipeptide repeat proteins (DPRs). As toxicity has been associated with both sense and antisense repeat-derived RNA and DPRs, targeting both strands may provide the most effective therapeutic strategy. CRISPR-Cas13 systems mature their own guide arrays, allowing targeting of multiple RNA species from a single construct. We show CRISPR-Cas13d variant CasRx effectively reduces overexpressed C9orf72 sense and antisense repeat transcripts and DPRs in HEK cells. In C9orf72 patient-derived iPSC-neuron lines, CRISPR-CasRx reduces endogenous sense and antisense repeat RNAs and DPRs and protects against glutamate-induced excitotoxicity. AAV delivery of CRISPR-CasRx to two distinct C9orf72 repeat mouse models significantly reduced both sense and antisense repeat-containing transcripts. This highlights the potential of RNA-targeting CRISPR systems as therapeutics for C9orf72 ALS/FTD.

Hydrogels can provide a hydrated environment to encapsulate extracellular vesicles (EVs) while offering promising solutions to some of the challenges that limit their therapeutic potential, e.g. rapid clearance and propensity for enzymatic degradation and aggregation. This study explores the use of a hyaluronic acid-tyramine (HA-TA) hydrogel to prolong the delivery and enhance the stability of EVs. EVs were obtained from lentiviral-transduced HEK293T cells expressing luciferase and eGFP to enable easy quantification. Two encapsulation strategies were evaluated: (1) pre-loading, where EVs were mixed with HA-TA (2.58 % degree of substitution) precursor solution and subsequently crosslinked with 2 U/mL horseradish peroxidase (HRP) and 0.05 mM H2O2; and (2) post-loading, where EVs were soaked into pre-formed dehydrated hydrogels. Both methods improved EV stability over 7 days at 37 °C compared to free EVs. The pre-loading approach was ultimately selected due to its ability to give rapid in situ gelation within one minute. Controlled in vitro release of EVs from the pre-loaded hydrogels was observed to extend beyond 7 days, as determined by CD9 ELISA. The released EVs maintained their bioactivity, as evidenced by effective internalisation into ARPE-19 and H9c2 cell lines, with performance comparable to fresh EVs. The EV release profile could be varied by modifying the hydrogel concentration. These findings underscore the potential of HA-TA hydrogels for localised, sustained, EV delivery with preserved functionality.

Movement disorders are a group of heterogeneous neurological conditions associated with alterations of tone, posture and voluntary movement. They may either occur in isolation or as part of a multisystemic condition. More recently, the advent of next generation sequencing technologies has facilitated better understanding of the underlying causative genes and molecular pathways, thereby identifying targets for genetic therapy. In this review, we summarize the advances in genetic therapy approaches for both hyperkinetic and hypokinetic movement disorders, including Parkinson’s Disease, Huntington’s Disease and rarer monogenic conditions of childhood onset. While there have been significant advances in the field, multiple challenges remain, related to safety, toxicity, efficacy and brain biodistribution, which will need to be addressed by the next generation of genetic therapies.

Background/Objectives: Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the GBA1 gene. Type 1 Gaucher disease is characterised by substrate accumulation in the visceral organs, which occurs in combination with acute and chronic neurodegeneration that distinguish type 2 and type 3 GD, respectively. We have previously shown the efficacy of neonatal AAV9 gene therapy for treating type 2 GD and aimed to investigate post-symptomatic administration into a model of type 1 disease. Current murine models of type 1 disease are limited in their recapitulation of early onset phenotypic manifestation and thus we aimed to create a novel model of type 1 in which to test the efficacy of adult gene therapy. Methods: The novel AAV-GD1 model was created through intracerebroventricular injection of AAV9 containing the human GBA1 gene under control of the neuron-specific synapsin promoter (AAV9.hSynI.hGBA1) to the pre-existing acute K14-lnl/lnl model of type 2 GD. Administration of AAV9.hSynI.hGBA1 aimed to restore glucocerebrosidase expression in the brain and extend the lifespan beyond 14 days, allowing the visceral pathology to develop further. The organ pathology was characterised by immunohistochemistry at various time points. Once visceral disease was confirmed, an intravenous injection of AAV9 containing a ubiquitously active CAG promoter driving hGBA1 (AAV9.CAG.hGBA1) was administered to post-symptomatic mice. Animals were aged for 2 and 4 months post-treatment with AAV9.CAG.hGBA1, and immunohistochemistry and enzymatic activity were assessed to investigate therapeutic efficacy. Results: The AAV-GD1 model displayed visceral pathology in the spleen, lung, and liver from 2 months of age. This allowed us to validate the efficacy of adult gene therapy; intravenous administration of AAV9.CAG.hGBA1 transiently ameliorated the lung pathology and rescued the spleen pathology up to 4 months post-administration. Conclusions: The creation of the novel AAV-GD1 model with more aggressive visceral pathology presents a unique opportunity for investigation of new therapies to treat type 1 GD. AAV9.CAG.hGBA1 represents a potential therapeutic option for all forms of Gaucher disease.

High ionic conductivity polymer electrolyte has been introduced to replace the liquid electrolyte used in commercial energy storage devices as they have a tendency in leakage. However, the high degree of crystallinity in polymer electrolyte limits further enhancement of ionic conductivity. In this work, a nanocomposite gel polymer electrolyte system comprising polymer blend of polyvinyl alcohol (PVA) - cellulose acetate (CA) with dopant lithium acetate (LiAc) salt in dimethylformamide (DMF) solvent has been prepared using solution casting method. Different concentrations of titanium dioxide (TiO2) 2 ) nanofiller were added to study the effect of nano- filler on characteristics of the electrolytes. Fourier transform infrared (FTIR) spectroscopy, impedance spectroscopy (EIS), and differential scanning calorimetry (DSC) were used to study the complexation between the materials, ionic conductivity, and thermal properties, respectively. The trend of the FTIR spectrum in the hydroxyl band showed that the lowered shifting wavenumber indicates a decreased crystalline phase with increasing TiO2 2 concentration. The highest conductivity of (3.30 +/- 0.15) x 10-- 4 S cm-- 1 at room temperature was obtained with addition of 8 wt% of TiO2. 2 . DSC analysis discovered the increase in conductivity is associated with a decrease in the glass transition temperature (Tg). Tg ). From transport number measurements (TNM), ions have been found to be the dominant charge carriers. Linear sweep voltammetry (LSV) result indicated that the most conducting electrolyte was electrochemically stable up to 2.2 V. The highest conducting electrolyte was used in the application of an electrochemical double layer capacitor (EDLC). The performance of the EDLC was characterized by cyclic voltammetry (CV) and galvanostatic charge-discharge (GCD) techniques. Specific capacitance (Csp) C sp ) of the electrode obtained from CV was 26.23 F g- 1 at 5 mV s-1 scan rate. The EDLC had been charged and discharged for 4600 cycles with the highest single electrode specific discharge capacitance (Cd) C d ) value of 18.01 F g- 1 .

Intensity modulated direct detection data transmission using microresonator multi-soliton and 2-soliton crystal states are demonstrated. We achieve error free transmission of 10Gb/s NRZ data and BERs in the region of 10-4 with 28.05GBd/s PAM4 data.

High ionic conductivity polymer electrolyte has been developed as a replacement for liquid electrolytes, which are prone to leaking when employed in dye-sensitized solar cells. However, the high degree of crystallinity in the polymer electrolyte hinders further ionic conductivity improvement. In this study, a gel polymer electrolyte was synthesized by stirring polyvinyl alcohol (PVA) with potassium iodide (KI) salt in dimethyl sulfoxide (DMSO), ethylene carbonate (EC), and propylene carbonate (PC). Different concentrations of diethyl carbonate (DEC) which acts as a plasticizer were added to study the effect of plasticizers on electrolyte conductivity. Impedance spectroscopy (EIS) and X-ray diffraction (XRD) were utilized to investigate the ionic conductivity and structural of the gel electrolyte. Based on the EIS, the optimum ionic conductivity of similar to 11 x 10(-3) S cm(-1) was acquired with addition of 25 wt.% of DEC. This study demonstrates that adding 25% wt.% DEC increased the gel electrolyte's ionic conductivity, suggesting that the plasticizer contributes to better ion mobility in the gel electrolyte's polymer matrix. XRD analysis revealed a decrease in crystallinity in the presence of DEC. The highest conducting electrolyte can serve as electrolyte material in the dye-sensitized solar cells to further study on their performance. In conclusion, DEC may improve the ionic conductivity as well as the amorphous characteristics of gel polymer electrolyte.

In addition to proteins, discussed in the Chapter "Advances in Vaccine Adjuvants: Nanomaterials and Small Molecules", there are a wide range of alternatives to small molecule active ingredients. Cells, extracellular vesicles, and nucleic acids in particular have attracted increasing research attention in recent years. There are now a number of products on the market based on these emerging technologies, the most famous of which are the mRNA-based vaccines against SARS-COV-2. These advanced therapeutic moieties are challenging to formulate however, and there remain significant challenges for their more widespread use. In this chapter, we consider the potential and bottlenecks for developing further medical products based on these systems. Cells, extracellular vesicles, and nucleic acids will be discussed in terms of their mechanism of action, the key requirements for translation, and how advanced formulation approaches can aid their future development. These points will be presented with selected examples from the literature, and with a focus on the formulations which have made the transition to clinical trials and clinical products.

We engineered HEK293T cells with a transgene encoding tetracycline-inducible expression of a Staphylococcus aureus nuclease incorporating a translocation signal. We adapted the unmodified and nuclease-engineered cell lines to grow in suspension in serum-free media, generating the HEK293TS and NuPro-2S cell lines, respectively. Transient transfection yielded 1.19 × 106 lentiviral transducing units per milliliter (TU/mL) from NuPro-2S cells and 1.45 × 106 TU/mL from HEK293TS cells. DNA ladder disappearance revealed medium-resident nuclease activity arising from NuPro-2S cells in a tetracycline-inducible manner. DNA impurity levels in lentiviral material arising from NuPro-2S and HEK293TS cells were undetectable by SYBR Safe agarose gel staining. Direct measurement by PicoGreen reagent revealed DNA to be present at 636 ng/mL in lentiviral material from HEK293TS cells, an impurity level reduced by 89% to 70 ng/mL in lentiviral material from NuPro-2S cells. This reduction was comparable to the 23 ng/mL achieved by treating HEK293TS-derived lentiviral material with 50 units/mL Benzonase.