Explore the vast range of titles available.

Prime editing enables precise installation of genomic substitutions, insertions and deletions in living systems. Efficient in vitro and in vivo delivery of prime editing components, however, remains a challenge. Here we report prime editor engineered virus-like particles (PE-eVLPs) that deliver prime editor proteins, prime editing guide RNAs and nicking single guide RNAs as transient ribonucleoprotein complexes. We systematically engineered v3 and v3b PE-eVLPs with 65- to 170-fold higher editing efficiency in human cells compared to a PE-eVLP construct based on our previously reported base editor eVLP architecture. In two mouse models of genetic blindness, single injections of v3 PE-eVLPs resulted in therapeutically relevant levels of prime editing in the retina, protein expression restoration and partial visual function rescue. Optimized PE-eVLPs support transient in vivo delivery of prime editor ribonucleoproteins, enhancing the potential safety of prime editing by reducing off-target editing and obviating the possibility of oncogenic transgene integration. Delivery of prime editors in vivo is improved using virus-like particles.

Viruses and virally derived particles have the intrinsic capacity to deliver molecules to cells, but the difficulty of readily altering cell-type selectivity has hindered their use for therapeutic delivery. Here, we show that cell surface marker recognition by antibody fragments displayed on membrane-derived particles encapsulating CRISPR–Cas9 protein and guide RNA can deliver genome editing tools to specific cells. Compared to conventional vectors like adeno-associated virus that rely on evolved capsid tropisms to deliver virally encoded cargo, these Cas9-packaging enveloped delivery vehicles (Cas9-EDVs) leverage predictable antibody–antigen interactions to transiently deliver genome editing machinery selectively to cells of interest. Antibody-targeted Cas9-EDVs preferentially confer genome editing in cognate target cells over bystander cells in mixed populations, both ex vivo and in vivo. By using multiplexed targeting molecules to direct delivery to human T cells, Cas9-EDVs enable the generation of genome-edited chimeric antigen receptor T cells in humanized mice, establishing a programmable delivery modality with the potential for widespread therapeutic utility. Cell-specific molecular delivery with enveloped delivery vehicles enables genome editing ex vivo and in vivo.

Chimeric antigen receptor (CAR)-T-cell therapy for solid tumors is limited due to heterogeneous target antigen expression and outgrowth of tumors lacking the antigen targeted by CAR-T cells directed against single antigens. Here, we developed a bicistronic construct to drive expression of a CAR specific for EGFRvIII, a glioblastoma-specific tumor antigen, and a bispecific T-cell engager (BiTE) against EGFR, an antigen frequently overexpressed in glioblastoma but also expressed in normal tissues. CART.BiTE cells secreted EGFR-specific BiTEs that redirect CAR-T cells and recruit untransduced bystander T cells against wild-type EGFR. EGFRvIII-specific CAR-T cells were unable to completely treat tumors with heterogenous EGFRvIII expression, leading to outgrowth of EGFRvIII-negative, EGFR-positive glioblastoma. However, CART.BiTE cells eliminated heterogenous tumors in mouse models of glioblastoma. BiTE-EGFR was locally effective but was not detected systemically after intracranial delivery of CART.BiTE cells. Unlike EGFR-specific CAR-T cells, CART.BiTE cells did not result in toxicity against human skin grafts in vivo. BiTE-secreting CAR-T cells overcome antigen escape from EGFRvIII-targeted therapy for glioblastoma.

Oral administration provides a simple and non-invasive approach for drug delivery. However, due to poor absorption and swift enzymatic degradation in the gastrointestinal tract, a wide range of molecules must be parenterally injected to attain required doses and pharmacokinetics. Here we present an orally dosed liquid auto-injector capable of delivering up to 4-mg doses of a bioavailable drug with the rapid pharmacokinetics of an injection, reaching an absolute bioavailability of up to 80% and a maximum plasma drug concentration within 30 min after dosing. This approach improves dosing efficiencies and pharmacokinetics an order of magnitude over our previously designed injector capsules and up to two orders of magnitude over clinically available and preclinical chemical permeation enhancement technologies. We administered the capsules to swine for delivery of clinically relevant doses of four commonly injected medications, including adalimumab, a GLP-1 analog, recombinant human insulin and epinephrine. These multi-day dosing experiments and oral administration in awake animal models support the translational potential of the system. Biologics are delivered by a pill that pricks the stomach wall.

The use of the edible photosynthetic cyanobacterium Arthrospira platensis (spirulina) as a biomanufacturing platform has been limited by a lack of genetic tools. Here we report genetic engineering methods for stable, high-level expression of bioactive proteins in spirulina, including large-scale, indoor cultivation and downstream processing methods. Following targeted integration of exogenous genes into the spirulina chromosome (chr), encoded protein biopharmaceuticals can represent as much as 15% of total biomass, require no purification before oral delivery and are stable without refrigeration and protected during gastric transit when encapsulated within dry spirulina. Oral delivery of a spirulina-expressed antibody targeting campylobacter—a major cause of infant mortality in the developing world—prevents disease in mice, and a phase 1 clinical trial demonstrated safety for human administration. Spirulina provides an advantageous system for the manufacture of orally delivered therapeutic proteins by combining the safety of a food-based production host with the accessible genetic manipulation and high productivity of microbial platforms. Spirulina is used to manufacture a therapeutic antibody against campylobacter.

Efficient protein delivery using cationic lipid transfection reagents enables high efficiency protein-based genome editing in vivo and in vitro . Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo , achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.

Key Points Biopharmaceutical drugs such as antibodies, peptides and recombinant proteins have high specificity and potency compared to small molecules. These features arise from their macromolecular composition, which provides the structural complexity that is often required for specificity. However, this structural complexity means that biopharmaceutical drugs are large and susceptible to degradation, which makes it challenging to formulate and deliver them. These drugs also have reduced permeation across biological barriers, which complicates their delivery to specific sites or intracellular targets. In this Review we highlight recent advances in formulation and delivery strategies that have facilitated the transformation of product portfolios and development pipelines by this class of compounds. These advances include the use of microsphere-based sustained-release technologies, protein modification methods that make use of polyethylene glycol and other polymers, as well as genetic manipulation of biopharmaceutical drugs such as Fc- and albumin-fusions. We also highlight current and emerging delivery routes that provide alternatives to injection, including transdermal, oral and pulmonary delivery. Current areas of formulation and delivery research show promise for the application of biopharmaceutical drugs to tumour immunotherapy using nanoparticle technology, tissue engineering and enhanced approaches to cell-based therapy. These delivery methods could be used for the targeted delivery of proteins to the brain, which could have implications in the treatment of a wide range of central nervous system disorders. These technologies could potentially increase the effectiveness of conventional approaches that have not yet translated to the clinic, although they have had promising preclinical results. Intracellular delivery of proteins and peptides is a new frontier in delivery research, which could dramatically augment the breadth of targets amenable to biopharmaceutical drug therapy. Biological drugs offer high specificity and potency, but their formulation and delivery pose substantial challenges. Here, the authors highlight recent advances in formulation strategies, describe current and emerging delivery routes and review the potential of targeted and intracellular delivery of biologics. The formulation and delivery of biopharmaceutical drugs, such as monoclonal antibodies and recombinant proteins, poses substantial challenges owing to their large size and susceptibility to degradation. In this Review we highlight recent advances in formulation and delivery strategies — such as the use of microsphere-based controlled-release technologies, protein modification methods that make use of polyethylene glycol and other polymers, and genetic manipulation of biopharmaceutical drugs — and discuss their advantages and limitations. We also highlight current and emerging delivery routes that provide an alternative to injection, including transdermal, oral and pulmonary delivery routes. In addition, the potential of targeted and intracellular protein delivery is discussed.

Epidermal growth factor is an excellent drug for promoting wound healing; however, its conventional administration strategies are associated with pharmacodynamic challenges, such as low transdermal permeability, reduction, and receptor desensitization. Here, we develop a microneedle-based self-powered transcutaneous electrical stimulation system (mn-STESS) by integrating a sliding free-standing triboelectric nanogenerator with a microneedle patch to achieve improved epidermal growth factor pharmacodynamics. We show that the mn-STESS facilitates drug penetration and utilization by using microneedles to pierce the stratum corneum. More importantly, we find that it converts the mechanical energy of finger sliding into electricity and mediates transcutaneous electrical stimulation through microneedles. We demonstrate that the electrical stimulation applied by mn-STESS acts as an “adjuvant” that suppresses the reduction of epidermal growth factor by glutathione and upregulates its receptor expression in keratinocyte cells, successfully compensating for receptor desensitization. Collectively, this work highlights the promise of self-powered electrical adjuvants in improving drug pharmacodynamics, creating combinatorial therapeutic strategies for traditional drugs. The use of epidermal growth factor for wound healing is limited by transdermal permeability, reduction, and receptor desensitization. Here the authors develop a microneedle-based self-powered transcutaneous electrical stimulation system to overcome these challenges.

Bacteria-based tumor therapy has recently attracted wide attentions due to its unique capability in targeting tumors and preferentially colonizing the core area of the tumor. Various therapeutic genes are also harbored into these engineering bacteria to enhance their anti-tumor efficacy. However, it is difficult to spatiotemporally control the expression of these inserted genes in the tumor site. Here, we engineer an ultrasound-responsive bacterium (URB) which can induce the expression of exogenous genes in an ultrasound-controllable manner. Owing to the advantage of ultrasound in tissue penetration, an acoustic remote control of bacterial gene expression can be realized by designing a temperature-actuated genetic switch. Cytokine interferon-γ (IFN-γ), an important immune regulatory molecule that plays a significant role in tumor immunotherapy, is used to test the system. Our results show that brief hyperthermia induced by focused ultrasound promotes the expression of IFN-γ gene, improving anti-tumor efficacy of URB in vitro and in vivo. Our study provides an alternative strategy for bacteria-mediated tumor immunotherapy. Several approaches have been recently proposed to engineer bacteria for cancer immunotherapy. Here the authors design an ultrasound-responsive bacterium for the controlled release of IFNy at the tumor site, promoting anti-tumor immune responses in preclinical models.

Peptides and proteins are widely used to treat a range of medical conditions; however, they often have to be injected and their effects are short-lived. These shortcomings of the native structure can be addressed by molecular engineering, but this is a complex undertaking. A molecular engineering technology initially applied to insulin — and which has now been successfully applied to several biopharmaceuticals — entails the derivatization of peptides and proteins with fatty acids. Various protraction mechanisms are enabled by the specific characteristics and positions of the attached fatty acid. Furthermore, the technology can ensure a long half-life following oral administration of peptide drugs, can alter the distribution of peptides and may hold potential for tissue targeting. Due to the inherent safety and well-defined chemical nature of the fatty acids, this technology provides a versatile approach to peptide and protein drug discovery.Peptide and protein drugs have proven successful in the treatment of a wide range of diseases, but their use can be limited by their inherent short-life and need for parenteral administration. Here, Kurtzhals et al. discuss how fatty acid derivatization can be applied to address these issues and optimize the pharmacological properties of peptide and protein drugs, highlighting associated considerations and future directions.