Nature Biotechnology
Nature Biotechnology is a monthly journal publishing new concepts in biological technology of relevance to bioengineering, medicine, energy, agriculture, food and the environment. It has a magazine covering the commercial, political, ethical, legal and societal aspects of this research.
A recent study found high amounts of microplastics in the human brain. This could spur funding and technological advances for plastic degradation.
Nature Biotechnology - Author Correction: Mapping multimodal phenotypes to perturbations in cells and tissue with CRISPRmap
Nature Biotechnology - Author Correction: Intravenous administration of blood–brain barrier-crossing conjugates facilitate biomacromolecule transport into central nervous system
DNA delivery using lipid nanoparticles results in severe toxicity in mice. However, we find that the incorporation of endogenous anti-inflammatory lipids into the lipid nanoparticles mitigates this toxicity and enables prolonged gene expression.
Nature Biotechnology - Author Correction: Multiplexed, image-based pooled screens in primary cells and tissues with PerturbView
Human lungs contain unique cell populations in distal respiratory airways or terminal and respiratory bronchioles (RA/TRBs) that accumulate in persons with lung injury and idiopathic pulmonary fibrosis (IPF), a lethal lung disease. As these populations are absent in rodents, deeper understanding requires a human in vitro model. Here we convert human pluripotent stem cells (hPS cells) into expandable spheres, called induced respiratory airway progenitors (iRAPs), consisting of ~98% RA/TRB-associated cell types. One hPS cell can give rise to 1010 iRAP cells. We differentiate iRAPs through a stage consistent with transitional type 2 alveolar epithelial (AT2) cells into a population corresponding to mature AT1 cells with 95% purity. iRAPs with deletion of Heřmanský–Pudlák Syndrome 1 (HPS1), which causes pulmonary fibrosis in humans, replicate the aberrant differentiation and recruitment of profibrotic fibroblasts observed in IPF, indicating that intrinsic dysfunction of RA/TRB-associated alveolar progenitors contributes to HPS1-related IPF. iRAPs may provide a system suitable for IPF drug discovery and validation. Human pluripotent cells are differentiated into lung progenitors and alveolar type 1 cells.
The mechanism of translation initiation in linear and circular mRNAs influences translation efficiency. Covalent attachment of an N7-methylguanosine (m7G) cap increases protein production from circular mRNAs in mice. Hybridization with capped endogenous RNAs also promotes protein production in cells, suggesting that this interaction might also occur between endogenous RNAs.
Circular mRNA faces challenges in enhancing its translation potential as an RNA therapeutic. Here we introduce two molecular designs that bolster circular mRNA translation through an internal cap-initiated mechanism. The first consists of a circular mRNA with a covalently attached N7-methylguanosine (m7G) cap through a branching structure (cap-circ mRNA). This modification allows circular mRNA to recruit translation machinery and produce proteins more efficiently than internal ribosome entry site (IRES)-containing circular mRNAs. Combining with an N1-methylpseudouridine (m1Ψ) modification, cap-circ mRNA exhibits a lower acute immunostimulatory effect, maintaining high translation in mice. The second design features the non-covalent attachment of an m7G cap to a circular mRNA through hybridization with an m7G cap-containing oligonucleotide, enhancing translation by more than 50-fold. This setup allows circular mRNAs to synthesize reporter proteins upon hybridizing with capped mRNAs or long non-coding RNAs and to undergo rolling circle-type translation. These advancements broaden the therapeutic applications of circular mRNAs by minimizing their molecular size, elevating translation efficiency and facilitating cell-type-selective translation. Placement of an m7G cap internally on circular RNAs promotes their translation in vivo.
Visualizing RNA molecules in live cells remains a challenge, and existing methods require genetic manipulation or have limited resolution. Our study overcomes these limitations by using the programmable CRISPR–Csm tool to bind and track individual transcripts in their native state.
Understanding the diverse dynamic behaviors of individual RNA molecules in single cells requires visualizing them at high resolution in real time. However, single-molecule live-cell imaging of unmodified endogenous RNA has not yet been achieved in a generalizable manner. Here, we present single-molecule live-cell fluorescence in situ hybridization (smLiveFISH), a robust approach that combines the programmable RNA-guided, RNA-targeting CRISPR–Csm complex with multiplexed guide RNAs for direct and efficient visualization of single RNA molecules in a range of cell types, including primary cells. Using smLiveFISH, we track individual native NOTCH2 and MAP1B transcripts in living cells and identify two distinct localization mechanisms including the cotranslational translocation of NOTCH2 mRNA at the endoplasmic reticulum and directional transport of MAP1B mRNA toward the cell periphery. This method has the potential to unlock principles governing the spatiotemporal organization of native transcripts in health and disease. Single-molecule live-cell fluorescence in situ hybridization uses an RNA-targeting CRISPR–Csm complex to image and track endogenous RNAs.
Oral administration of biologic drugs is challenging because of the degradative activity of the upper gastrointestinal tract. Strategies that use engineered microbes to produce biologics in the lower gastrointestinal tract are limited by competition with resident commensal bacteria. Here we demonstrate the engineering of bacteriophage (phage) that infect resident commensals to express heterologous proteins released during cell lysis. Working with the virulent T4 phage, which targets resident, nonpathogenic Escherichia coli, we first identify T4-specific promoters with maximal protein expression and minimal impact on T4 phage titers. We engineer T4 phage to express a serine protease inhibitor of a pro-inflammatory enzyme with increased activity in ulcerative colitis and observe reduced enzyme activity in a mouse model of colitis. We also apply the approach to reduce weight gain and inflammation in mouse models of diet-induced obesity. This work highlights an application of virulent phages in the mammalian gut as engineerable vectors to release therapeutics from resident gut bacteria. Biologics are delivered to the gut using phage that infects resident commensal bacteria.
Society, patients and clinicians welcome a much-needed non-opioid pain medication. Vertex’s first-in-class analgesic Journavx could soon be followed by a new generation of addiction-free pain drugs acting at NaV1.8 sodium channels.
Reverse transcriptase (RT) has been shown to play a role in double-strand break repair in bacteria, yet the impact of the RT component of prime editors (PEs) on normal mammalian cellular functions is unclear. Here we show that overexpressed RT or PE increases short insertions and diminishes homology-directed repair following Cas9 cleavage at multiple loci in multiple cell lines. Live-cell imaging shows that RT and PEs are rapidly recruited to laser-induced DNA damage sites and promote endogenous repair, independent of known DNA damage sensors. Interestingly, RT–mCherry partially impairs green fluorescent protein–PARP1 recruitment. A compact PE without an RNase H domain shows reduced DNA repair activity and may therefore be more suitable for clinical application. These data reveal a role for untethered RT or the RT domain of PEs in the repair of chromosomal breaks, calling for evaluation of the long-term effect of PEs and retroviral RT in mammalian cells. DNA repair activity of reverse transcriptase may negatively affect prime editing precision.
Recent patents relating to antibody compositions and methods of use.
Nature Biotechnology - Biotech news from around the world
Nature Biotechnology - Heat-resistant rice without yield compromise
Nature Biotechnology - Gene and cell therapies for Parkinson’s make headway
Nature Biotechnology - Largest proteome study enlists 14 biopharmas
Nature Biotechnology - De novo-designed protein binders neutralize snake toxins
Nature Biotechnology - Startup grows egg proteins in potato fields
Nature Biotechnology - A chromatin-based model for deciphering gene interactions
The first clinical results in patients with a genetic form of frontotemporal dementia (FTD) show that enhancing progranulin in the brain may halt disease progression. If successful, this potentially disease-modifying approach may uncover new avenues for treating other neurodegenerative diseases.
The possibility of collateral RNA degradation poses a concern for transcriptome perturbations and therapeutic applications using CRISPR–Cas13. We show that collateral activity only occurs with high RfxCas13d expression. Using low-copy RfxCas13d in transcriptome-scale and combinatorial pooled screens, we achieve high on-target knockdown without extensive collateral activity. Furthermore, analysis of a high-fidelity Cas13 variant suggests that its reduced collateral activity may be due to overall diminished nuclease capability. Careful selection of Cas13 variants and delivery methods minimizes collateral RNA degradation.
Engineering T cell specificity and function at multiple loci can generate more effective cellular therapies, but current manufacturing methods produce heterogenous mixtures of partially engineered cells. Here we develop a one-step process to enrich unlabeled cells containing knock-ins at multiple target loci using a family of repair templates named synthetic exon expression disruptors (SEEDs). SEEDs associate transgene integration with the disruption of a paired target endogenous surface protein while preserving target expression in nonmodified and partially edited cells to enable their removal (SEED-Selection). We design SEEDs to modify three critical loci encoding T cell specificity, coreceptor expression and major histocompatibility complex expression. The results demonstrate up to 98% purity after selection for individual modifications and up to 90% purity for six simultaneous edits (three knock-ins and three knockouts). This method is compatible with existing clinical manufacturing workflows and can be readily adapted to other loci to facilitate production of complex gene-edited cell therapies. Primary T cells with multiple genetic modifications are rapidly isolated by negative selection.
The value of lipid nanoparticles (LNPs) for delivery of messenger RNA (mRNA) was demonstrated by the coronavirus disease 2019 (COVID-19) mRNA vaccines, but the ability to use LNPs to deliver plasmid DNA (pDNA) would provide additional advantages, such as longer-term expression and availability of promoter sequences. However, pDNA-LNPs face substantial challenges, such as toxicity and low delivery efficiency. Here we show that pDNA-LNPs induce acute inflammation in naive mice that is primarily driven by the cGAS–STING pathway. Inspired by DNA viruses that inhibit this pathway for replication, we loaded endogenous lipids that inhibit STING into pDNA-LNPs. Loading nitro-oleic acid (NOA) into pDNA-LNPs (NOA-pDNA-LNPs) ameliorated serious inflammatory responses in vivo, enabling safer, prolonged transgene expression—11.5 times greater than that of mRNA-LNPs at day 32. Additionally, we performed a small LNP formulation screen to iteratively optimize transgene expression and increase expression 50-fold in vitro. pDNA-LNPs loaded with NOA and other bioactive molecules should advance genetic medicine by enabling longer-term and promoter-controlled transgene expression. Lipid nanoparticles carrying plasmid DNA and an inflammation inhibitor enable longer transgene expression than mRNA nanoparticles.
Adipocytes can be isolated, genetically manipulated, and then reimplanted. In this study, these properties were leveraged to engineer adipocytes that can outcompete tumors for essential metabolic resources. In mouse cancer models, these adipocytes suppressed tumor growth, demonstrating a novel cancer therapy termed adipose manipulation transplantation (AMT).
Ultrasound neurotechnologies are moving quickly into clinical trials in a wide variety of applications, and initiatives to open-source their manufacture will make them more accessible.
Tumors exhibit an increased ability to obtain and metabolize nutrients. Here, we implant engineered adipocytes that outcompete tumors for nutrients and show that they can substantially reduce cancer progression, a technology termed adipose manipulation transplantation (AMT). Adipocytes engineered to use increased amounts of glucose and fatty acids by upregulating UCP1 were placed alongside cancer cells or xenografts, leading to significant cancer suppression. Transplanting modulated adipose organoids in pancreatic or breast cancer genetic mouse models suppressed their growth and decreased angiogenesis and hypoxia. Co-culturing patient-derived engineered adipocytes with tumor organoids from dissected human breast cancers significantly suppressed cancer progression and proliferation. In addition, cancer growth was impaired by inducing engineered adipose organoids to outcompete tumors using tetracycline or placing them in an integrated cell-scaffold delivery platform and implanting them next to the tumor. Finally, we show that upregulating UPP1 in adipose organoids can outcompete a uridine-dependent pancreatic ductal adenocarcinoma for uridine and suppress its growth, demonstrating the potential customization of AMT. Adipose manipulation transplantation can reduce tumor growth and proliferation in vitro and in mouse models.
Here we report a method, smol-seq (small-molecule sequencing), using structure-switching aptamers (SSAs) and DNA sequencing to quantify metabolites. In smol-seq, each SSA detects a single target molecule and releases a unique DNA barcode on target binding. Sequencing the released barcodes can, thus, read out metabolite levels. We show that SSAs are highly specific and can be multiplexed to detect multiple targets in parallel, bringing the power of DNA sequencing to metabolomics. Metabolites can be quantified using a combination of aptamers and DNA barcodes.
Nature Biotechnology - Author Correction: Multiplexed inhibition of immunosuppressive genes with Cas13d for combinatorial cancer immunotherapy
Nature Biotechnology - Author Correction: Bioinstructive implantable scaffolds for rapid in vivo manufacture and release of CAR-T cells
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