Focused ultrasound mitigates pathology and improves spatial memory in Alzheimer's mice and patients

Rationale: Bilateral sonication with focused ultrasound (FUS) in conjunction with microbubbles has been shown to separately reduce amyloid plaques and hyperphosphorylated tau protein in the hippocampal formation and the entorhinal cortex in different mouse models of Alzheimer's disease (AD) without any therapeutic agents. However, the two pathologies are expressed concurrently in human disease. Therefore, the objective of this study is to investigate the effects of repeated bilateral sonications in the presence of both pathologies. Methods: Herein, we investigate its functional and morphological outcomes on brains bearing both pathologies simultaneously. Eleven transgenic mice of the 3xTg-AD line (14 months old) expressing human amyloid beta and human tau and eleven age-matched wild-type littermates received four weekly bilateral sonications covering the hippocampus followed by working memory testing. Afterwards, immunohistochemistry and immunoassays (western blot and ELISA) were employed to assess any changes in amyloid beta and human tau. Furthermore, we present preliminary data from our clinical trial using a neuronavigation-guided FUS system for sonications in AD patients (NCT04118764). Results: Interestingly, both wild-type and transgenic animals that received FUS experienced improved working memory and spent significantly more time in the escape platform-quadrant, with wild-type animals spending 43.2% (sham: 37.7%) and transgenic animals spending 35.3% (sham: 31.0%) of the trial in the target quadrant. Furthermore, this behavioral amelioration in the transgenic animals correlated with a 58.3% decrease in the neuronal length affected by tau and a 27.2% reduction in total tau levels. Amyloid plaque population, volume and overall load were also reduced overall. Consistently, preliminary data from a clinical trial involving AD patients showed a 1.8% decrease of amyloid PET signal 3-weeks after treatment in the treated hemisphere compared to baseline. Conclusion: For the first time, it is shown that bilateral FUS-induced BBB opening significantly and simultaneously ameliorates both coexistent pathologies, which translated to improvements in spatial memory of transgenic animals with complex AD, the human mimicking phenotype. The level of cognitive improvement was significantly correlated with the volume of BBB opening. Non-transgenic animals were also shown to exhibit similar memory amelioration for the first time, indicating that BBB opening results into benefits in the neuronal function regardless of the existence of AD pathology. A potential mechanism of action for the reduction of the both pathologies investigated was the cholesterol metabolism, specifically the LRP1b receptor, which exhibited increased expression levels in transgenic mice following FUS-induced BBB opening. Initial clinical evidence supported that the beta amyloid reduction shown in rodents could be translatable to humans with significant amyloid reduction shown in the treated hemisphere

Nonviral gene editing via CRISPR/Cas9 delivery by membrane-disruptive and endosomolytic helical polypeptide

Effective and safe delivery of the CRISPR/Cas9 gene-editing elements remains a challenge. Here we report the development of PEGylated nanoparticles (named P-HNPs) based on the cationic α-helical polypeptide poly(γ-4-((2-(piperidin-1-yl)ethyl)aminomethyl)benzyl-L-glutamate) for the delivery of Cas9 expression plasmid and sgRNA to various cell types and gene-editing scenarios. The cell-penetrating α-helical polypeptide enhanced cellular uptake and promoted escape of pCas9 and/or sgRNA from the endosome and transport into the nucleus. The colloidally stable P-HNPs achieved a Cas9 transfection efficiency up to 60% and sgRNA uptake efficiency of 67.4%, representing an improvement over existing polycation-based gene delivery systems. After performing single or multiplex gene editing with an efficiency up to 47.3% in vitro, we demonstrated that P-HNPs delivering Cas9 plasmid/sgRNA targeting the polo-like kinase 1 (Plk1) gene achieved 35% gene deletion in HeLa tumor tissue to reduce the Plk1 protein level by 66.7%, thereby suppressing the tumor growth by >71% and prolonging the animal survival rate to 60% within 60 days. Capable of delivering Cas9 plasmids to various cell types to achieve multiplex gene knock-out, gene knock-in, and gene activation in vitro and in vivo, the P-HNP system offers a versatile gene-editing platform for biological research and therapeutic applications

Focused ultrasound-mediated brain genome editing.

Gene editing in the brain has been challenging because of the restricted transport imposed by the blood-brain barrier (BBB). Current approaches mainly rely on local injection to bypass the BBB. However, such administration is highly invasive and not amenable to treating certain delicate regions of the brain. We demonstrate a safe and effective gene editing technique by using focused ultrasound (FUS) to transiently open the BBB for the transport of intravenously delivered CRISPR/Cas9 machinery to the brain

Nanosystems for Gene Editing and Targeted Therapy

Nanomedicine has emerged in the past decades, and a variety of designs for drug/gene delivery have been reported since the concept of nanomedicine was first demonstrated. However, with the exception of a few notable successes, the clinical translation of nanomedicine has been slow. Specificity and delivery efficiency are the major obstacles; only a few nanomedicine systems can effectively reach and release the therapeutic payload at the target site, thereby limiting the therapeutic efficacy. To tackle these issues, this work aims to design new strategies to improve nanomedicine systems at the gene-, protein- and tissue- levels. We applied CRISPR/Cas9 technology for gene targeting. Delivering CRISPR/Cas9 elements, including Cas9 endonuclease and a corresponding guide RNA, allows for specific gene mutagenesis. A conventional gene delivery carrier often has a highly positive charge density for higher transgene expression, but this may result in unfavorable effects on the Cas9 plasmid transfection. As a large plasmid, strong interaction between the Cas9 plasmid and the polycation with high charge density may hinder the plasmid’s intracellular release. Moreover, high Cas9 expression usually leads to undesirable off-target effects. We addressed these two major obstacles by designing a low-charged density micelle, composed of quaternary ammonium‐terminated poly(propylene oxide) and amphiphilic Pluronic F127. We tested this design on a human papillomavirus (HPV)-induced cervical cancer model to target the HPV oncogene, E7. Our micellar carrier enabled effective Cas9 transfection with a transient Cas9 expression, which offered enhanced Cas9 on-target specificity. This nonviral Cas9‐mediated E7 mutagenesis resulted in significant inhibition of HPV‐induced cancerous activity both in vitro and in vivo. Although CRISPR/Cas9 technology is a powerful toolkit for gene manipulation, gene editing might not be practical for therapeutics in the cancers that develop from endogenous mutations, which may vary among patients and disease stages. Protein-targeting, therefore, may be a more efficient approach. Aptamer and its selection technology, namely SELEX, offer direct evolution to obtain a nucleic acid ligand that specifically recognizes the protein target. Yet, aptamer screening remains unsatisfactory, and the success rate of SELEX is limited. We designed two approaches to improve the aptamer screening. We first employed a microarray platform to deconvolute the aptamer sequence and identified the aptamer functional motif. The resulted protein-targeting motif with an optimal length and showed enhanced structural and functional characteristics compared with its parental sequence. In addition to sequence optimization, conjunction of two distinct aptamers that recognize different epitopes of the protein target is another approach to improve the aptamer’s affinity. In looking for a rapid way to screen this bivalent aptamer pair, we designed a quantum dot (QD)/ Förster resonance energy transfer (FRET) sensor. Using a thrombin aptamer as a model system, we conjugated an anti-thrombin aptamer with QD and stained the other one with the intercalation dye, YOYO-3. If the two aptamers recognized different epitopes of thrombin, the conformational change of the two aptamers would take place when interacting with thrombin, and this would induce YOYO-3 dye’s translocation. YOYO-3 would be transferred from the aptamer to QD surface, resulting in a strong FRET signal. In contrast, if they recognized the same epitope, binding competition between two aptamers would inhibit dye translocation, thereby giving a minimal FRET signal. By measuring the FRET signal, we can verify if the two aptamers may form a bivalent pair. Lastly, we integrated mesenchymal stem cell (MSC) with a nanomedicine system to achieve active tissue-targeting. MSC is known to migrate toward certain types of cancer cells by chasing the chemotaxis release from the cancer cells, but the therapeutic payload that MSC can carry is limited. Forming an MSC spheroid allowed the loading of the nanomedicine system with another type of anti-cancer drug. We therefore designed a hybrid MSC/nanomedicine spheroid, which functioned as an active tumor-targeting platform, enabling effective delivery for both cytotoxic protein and chemotherapeutic drugs. In a heterotopic glioblastoma model, the hybrid spheroid significantly improved the retention of the nanomedicine system at the tumor site, leading to enhanced tumor inhibition in vivo. Collectively, this work demonstrated the effective approaches for gene, protein and tissue targeting by addressing the issues of low specificity and limited delivery efficiency that many current nanomedicine systems face. Particularly, the results may add to the armamentarium of cancer therapeutics, which remains largely challenging and intractable

Signal-on Protein Detection via Dye Translocation between Aptamer and Quantum Dot

A unique interaction between the cyanine dye and negatively charged quantum dot is used to construct a signal-on biaptameric quantum dot (QD) Förster resonance energy transfer (FRET) beacon for protein detection and distinct aptamer characterization. The beacon comprises a pair of aptamers, one intercalated with the cyanine dye (YOYO-3) and the other conjugated to a negatively charged, carboxyl-QD. When the target protein is present, structural folding and sandwich association of the two aptamers take place. As a consequence, YOYO-3 is displaced from the folded aptamer and transferred to the unblocked QD surface to yield a target concentration-dependent FRET signal. As a proof-of-principle, we demonstrate the detection of thrombin ranging from nanomolar to submicromolar concentrations and confirm the dye translocation using cylindrical illumination confocal spectroscopy (CICS). The proposed beacon provides a simple, rapid, signal-on FRET detection for protein as well as a potential platform for distinct aptamer screening

Phenylboronic Acid-Functionalized Polyplexes Tailored to Oral CRISPR Delivery

Effective delivery of the CRISPR-Cas9 components is crucial to realizing the therapeutic potential. Although many delivery approaches have been developed for this application, oral delivery has not been explored due to the degradative nature of the gastrointestinal tract. For this issue, we developed a series of novel phenylboronic acid (PBA)-functionalized chitosan-polyethylenimine (CS-PEI) polymers for oral CRISPR delivery. PBA functionalization equipped the polyplex with higher stability, smooth transport across the mucus, and efficient endosomal escape and cytosolic unpackaging in the cells. From a library of 12 PBA-functionalized CS-PEI polyplexes, we identified a formulation that showed the most effective penetration in the intestinal mucosa after oral gavage to mice. The optimized formulation performed feasible CRISPR-mediated downregulation of the target protein and reduction in the downstream cholesterol. As the first oral CRISPR carrier, this study suggests the potential of addressing the needs of both local and systemic editing in a patient-compliant manner