Transthyretin amyloidosis, also called ATTR amyloidosis, is a life-threatening disease characterized by progressive accumulation of misfolded transthyretin (TTR) protein in tissues, predominantly the ...nerves and heart. NTLA-2001 is an in vivo gene-editing therapeutic agent that is designed to treat ATTR amyloidosis by reducing the concentration of TTR in serum. It is based on the clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease (CRISPR-Cas9) system and comprises a lipid nanoparticle encapsulating messenger RNA for Cas9 protein and a single guide RNA targeting
.
After conducting preclinical in vitro and in vivo studies, we evaluated the safety and pharmacodynamic effects of single escalating doses of NTLA-2001 in six patients with hereditary ATTR amyloidosis with polyneuropathy, three in each of the two initial dose groups (0.1 mg per kilogram and 0.3 mg per kilogram), within an ongoing phase 1 clinical study.
Preclinical studies showed durable knockout of
after a single dose. Serial assessments of safety during the first 28 days after infusion in patients revealed few adverse events, and those that did occur were mild in grade. Dose-dependent pharmacodynamic effects were observed. At day 28, the mean reduction from baseline in serum TTR protein concentration was 52% (range, 47 to 56) in the group that received a dose of 0.1 mg per kilogram and was 87% (range, 80 to 96) in the group that received a dose of 0.3 mg per kilogram.
In a small group of patients with hereditary ATTR amyloidosis with polyneuropathy, administration of NTLA-2001 was associated with only mild adverse events and led to decreases in serum TTR protein concentrations through targeted knockout of
. (Funded by Intellia Therapeutics and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT04601051.).
•Graphene-based nanomaterials are emerging as the next generation’s candidates for biotechnological advancements.•Graphene and its various derivatives have been covered.•The properties of ...graphene-based materials related to biomedical applications have been discussed.•The review emphasizes the up-to-date biomedical applications of graphene-based materials.•It also covers the rising concern about the toxicity of graphene-based materials.
Here, we discuss the biomedical applications of graphene-based nanomaterials (GBNs). We examine graphene and its various derivatives, including graphene, graphene oxides (GOs), reduced graphene oxides (rGOs), graphene quantum dots (GQDs), and graphene composites, and discuss their unique properties related to their biomedical applications. We also summarize the detailed biomedical applications of GBNs, including drug and/or gene delivery, bioimaging, and tissue engineering. We also highlight the toxicity of these nanomaterials.
Efficient retrograde access to projection neurons for the delivery of sensors and effectors constitutes an important and enabling capability for neural circuit dissection. Such an approach would also ...be useful for gene therapy, including the treatment of neurodegenerative disorders characterized by pathological spread through functionally connected and highly distributed networks. Viral vectors, in particular, are powerful gene delivery vehicles for the nervous system, but all available tools suffer from inefficient retrograde transport or limited clinical potential. To address this need, we applied in vivo directed evolution to engineer potent retrograde functionality into the capsid of adeno-associated virus (AAV), a vector that has shown promise in neuroscience research and the clinic. A newly evolved variant, rAAV2-retro, permits robust retrograde access to projection neurons with efficiency comparable to classical synthetic retrograde tracers and enables sufficient sensor/effector expression for functional circuit interrogation and in vivo genome editing in targeted neuronal populations.
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•AAV can be endowed with robust retrograde functionality through directed evolution•Up to two orders of magnitude increase in retrograde transport over existing variants•Efficiency comparable to synthetic tracers•Sufficient payload expression for circuit interrogation and gene manipulation
Projection neurons are a critical component of large-scale networks distributing the results of local circuit computations between distant brain regions, but their specific contribution is often hard to pinpoint because of the difficulty of gaining selective genetic access. Tervo et al. introduce a designer variant of adeno-associated virus, rAAV2-retro, that allows for efficient mapping, monitoring, and manipulation of projection neurons.
Mutations in the gene encoding dystrophin, a protein that maintains muscle integrity and function, cause Duchenne muscular dystrophy (DMD). The deltaE50-MD dog model of DMD harbors a mutation ...corresponding to a mutational "hotspot" in the human
gene. We used adeno-associated viruses to deliver CRISPR gene editing components to four dogs and examined dystrophin protein expression 6 weeks after intramuscular delivery (
= 2) or 8 weeks after systemic delivery (
= 2). After systemic delivery in skeletal muscle, dystrophin was restored to levels ranging from 3 to 90% of normal, depending on muscle type. In cardiac muscle, dystrophin levels in the dog receiving the highest dose reached 92% of normal. The treated dogs also showed improved muscle histology. These large-animal data support the concept that, with further development, gene editing approaches may prove clinically useful for the treatment of DMD.
Delivery remains a major obstacle restricting the potential action of small molecular drugs as well as novel biologics which cannot readily enter cells without the help of a vector. A successful ...active delivery process involves three steps: (a) tagging the drug with a vector, (b) effective trafficking of this drug-vector conjugate through biological barriers, and finally (c) controlled drug release. While covalent bond formation and/or supramolecular association is involved in the making of the drug-vector conjugate, the final step requires precisely a controlled dissociation in order to trigger drug release. Therefore, in pursuit of smart, effective, and nontoxic delivery systems, it has become widely recognized that control over dynamic self-assembly could unleash the efficacy of artificial vectors. In this Account, I discuss our endeavors, and those of colleagues, in the recent implementation of Dynamic Covalent Chemistry (DCvC) in delivery applications. DCvC exploits reversible covalent reactions to generate covalent systems that can self-fabricate, adapt, respond, and fall apart in a controlled fashion. A privileged set of reversible covalent reactions has emerged in the community working on delivery applications and is based on condensation reactions (imine, acylhydrazone, oxime), and disulfide and boronate ester formations. The latest developments making this chemistry particularly attractive for such a DCvC approach are discussed. The rational justifying the potential of DCvC in delivery is based on the principle that using such reversible covalent reactions afford transient drug-vector conjugates which form spontaneously and chemoselectively, then adapt and self-correct their structure during self-assembly and trafficking thanks to the dynamic nature of the reversible covalent bonds, and finally respond to physicochemical stimuli such as pH and redox changes, thereby enabling controlled dissociation and concomitant drug release. For these reasons, DCvC has recently emerged as a leverage tool with growing prospects for advancing toward smarter delivery systems. The implementation of DCvC can follow three approaches that are discussed herein: (1) dynamic covalent bioconjugates, involving the transient covalent conjugation with a vector, (2) dynamic covalent vectors, involving the controlled dynamic and adaptive assembly and disassembly of vectors that complex drugs through supramolecular association, and (3) dynamic covalent targeting, involving the transient chemoselective formation of covalent bonds with the constituents of cell membranes. While DCvC has already attracted interest in material sciences, the recent results described in this Account showcase the vast potential of DCvC in biological sciences, and in particular in delivery applications where self-fabricated, adaptive, and responsive devices are of utmost importance.
Polymers represent a promising therapeutic platform for extrahepatic messenger RNA (mRNA) delivery but are hampered by low
efficacy due to polyplex serum instability and inadequate endosomal escape ...following systemic administration. Here, we report the rational design and combinatorial synthesis of zwitterionic phospholipidated polymers (ZPPs) via cationic polymer postmodification by alkylated dioxaphospholane oxides to deliver mRNA to spleen and lymph nodes
. This modular postmodification approach readily produces tunable zwitterionic species for serum resistance and introduces alkyl chains simultaneously to enhance endosomal escape, thereby transforming deficient cationic polymers to efficacious zwitterionic mRNA carriers without the need to elaborately synthesize functional monomers. ZPPs mediated up to 39 500-fold higher protein expression than their parent cationic counterparts
and enabled efficacious mRNA delivery selectively in spleen and lymph nodes following intravenous administration
. This zwitterionic phospholipidation methodology provides a versatile and generalizable postmodification strategy to introduce zwitterions into the side chains of cationic polymers, extending the utility of cationic polymer families for precise mRNA delivery and demonstrating substantial potential for immunotherapeutic applications.
Micro-dystrophin gene transfer shows promise for treating patients with Duchenne muscular dystrophy (DMD) using recombinant adeno-associated virus serotype rh74 (rAAVrh74) and codon-optimized human ...micro-dystrophin driven by a skeletal and cardiac muscle-specific promoter with enhanced cardiac expression (MHCK7).
To identify the 1-year safety and tolerability of intravenous rAAVrh74.MHCK7.micro-dystrophin in patients with DMD.
This open-label, phase 1/2a nonrandomized controlled trial was conducted at the Nationwide Children's Hospital in Columbus, Ohio. It began on November 2, 2017, with a planned duration of follow-up of 3 years, ending in March 2021. The first 4 patients who met eligibility criteria were enrolled, consisting of ambulatory male children with DMD without preexisting AAVrh74 antibodies and a stable corticosteroid dose (≥12 weeks).
A single dose of 2.0 × 1014 vg/kg rAAVrh74.MHCK7.micro-dystrophin was infused through a peripheral limb vein. Daily prednisolone, 1 mg/kg, started 1 day before gene delivery (30-day taper after infusion).
Safety was the primary outcome. Secondary outcomes included micro-dystrophin expression by Western blot and immunohistochemistry. Functional outcomes measured by North Star Ambulatory Assessment (NSAA) and serum creatine kinase were exploratory outcomes.
Four patients were included (mean SD age at enrollment, 4.8 1.0 years). All adverse events (n = 53) were considered mild (33 62%) or moderate (20 38%), and no serious adverse events occurred. Eighteen adverse events were considered treatment related, the most common of which was vomiting (9 of 18 events 50%). Three patients had transiently elevated γ-glutamyltransferase, which resolved with corticosteroids. At 12 weeks, immunohistochemistry of gastrocnemius muscle biopsy specimens revealed robust transgene expression in all patients, with a mean of 81.2% of muscle fibers expressing micro-dystrophin with a mean intensity of 96% at the sarcolemma. Western blot showed a mean expression of 74.3% without fat or fibrosis adjustment and 95.8% with adjustment. All patients had confirmed vector transduction and showed functional improvement of NSAA scores and reduced creatine kinase levels (posttreatment vs baseline) that were maintained for 1 year.
This trial showed rAAVrh74.MHCK7.micro-dystrophin to be well tolerated and have minimal adverse events; the safe delivery of micro-dystrophin transgene; the robust expression and correct localization of micro-dystrophin protein; and improvements in creatine kinase levels and NSAA scores. These findings suggest that rAAVrh74.MHCK7.micro-dystrophin can provide functional improvement that is greater than that observed under standard of care.
ClinicalTrials.gov Identifier: NCT03375164.
Self-assembled mRNA vaccines Kim, Jeonghwan; Eygeris, Yulia; Gupta, Mohit ...
Advanced drug delivery reviews,
03/2021, Letnik:
170
Journal Article
Recenzirano
Odprti dostop
mRNA vaccines have evolved from being a mere curiosity to emerging as COVID-19 vaccine front-runners. Recent advancements in the field of RNA technology, vaccinology, and nanotechnology have ...generated interest in delivering safe and effective mRNA therapeutics. In this review, we discuss design and self-assembly of mRNA vaccines. Self-assembly, a spontaneous organization of individual molecules, allows for design of nanoparticles with customizable properties. We highlight the materials commonly utilized to deliver mRNA, their physicochemical characteristics, and other relevant considerations, such as mRNA optimization, routes of administration, cellular fate, and immune activation, that are important for successful mRNA vaccination. We also examine the COVID-19 mRNA vaccines currently in clinical trials. mRNA vaccines are ready for the clinic, showing tremendous promise in the COVID-19 vaccine race, and have pushed the boundaries of gene therapy.
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•mRNA vaccines showed spectacular success in the race for COVID-19 vaccines.•Design of both mRNA and the delivery vectors help in fine-tuning efficacy.•Self-assembled mRNA delivery vectors include lipid and polymer nanoparticles.•Lipid nanoparticle mRNA vaccines have ca. 95% efficacy and earned EUA FDA approval.•These unprecedented results may pave the road for future mRNA vaccine development.
Non-viral vectors for gene-based therapy Yin, Hao; Kanasty, Rosemary L; Eltoukhy, Ahmed A ...
Nature reviews. Genetics,
08/2014, Letnik:
15, Številka:
8
Journal Article
Recenzirano
Gene-based therapy is the intentional modulation of gene expression in specific cells to treat pathological conditions. This modulation is accomplished by introducing exogenous nucleic acids such as ...DNA, mRNA, small interfering RNA (siRNA), microRNA (miRNA) or antisense oligonucleotides. Given the large size and the negative charge of these macromolecules, their delivery is typically mediated by carriers or vectors. In this Review, we introduce the biological barriers to gene delivery in vivo and discuss recent advances in material sciences, nanotechnology and nucleic acid chemistry that have yielded promising non-viral delivery systems, some of which are currently undergoing testing in clinical trials. The diversity of these systems highlights the recent progress of gene-based therapy using non-viral approaches.
Genome editing offers promising solutions to genetic disorders by editing DNA sequences or modulating gene expression. The clustered regularly interspaced short palindromic repeats ...(CRISPR)/associated protein 9 (CRISPR/Cas9) technology can be used to edit single or multiple genes in a wide variety of cell types and organisms in vitro and in vivo. Herein, we review the rapidly developing CRISPR/Cas9-based technologies for disease modeling and gene correction and recent progress toward Cas9/guide RNA (gRNA) delivery based on viral and nonviral vectors. We discuss the relative merits of delivering the genome editing elements in the form of DNA, mRNA, or protein, and the opportunities of combining viral delivery of a transgene encoding Cas9 with nonviral delivery of gRNA. We highlight the lessons learned from nonviral gene delivery in the past three decades and consider their applicability for CRISPR/Cas9 delivery. We also include a discussion of bioinformatics tools for gRNA design and chemical modifications of gRNA. Finally, we consider the extracellular and intracellular barriers to nonviral CRISPR/Cas9 delivery and propose strategies that may overcome these barriers to realize the clinical potential of CRISPR/Cas9-based genome editing.
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