Optimization strategies and advances in the research and development of AAV-based gene therapy to deliver large transgenes.

Adeno-associated virus (AAV)-based therapies are recognized as one of the most potent next-generation treatments for inherited and genetic diseases. However, several biological and technological aspects of AAV vectors remain a critical issue for their widespread clinical application. Among them, the limited capacity of the AAV genome significantly hinders the development of AAV-based gene therapy. In this context, genetically modified transgenes compatible with AAV are opening up new opportunities for unlimited gene therapies for many genetic disorders. Recent advances in de novo protein design and remodelling are paving the way for new, more efficient and targeted gene therapeutics. Using computational and genetic tools, AAV expression cassette and transgenic DNA can be split, miniaturized, shuffled or created from scratch to mediate efficient gene transfer into targeted cells. In this review, we highlight recent advances in AAV-based gene therapy with a focus on its use in translational research. We summarize recent research and development in gene therapy, with an emphasis on large transgenes (>4.8 kb) and optimizing strategies applied by biomedical companies in the research pipeline. We critically discuss the prospects for AAV-based treatment and some emerging challenges. We anticipate that the continued development of novel computational tools will lead to rapid advances in basic gene therapy research and translational studies.

Multiplexed CRISPR-Cas9 protocol for large transgene integration into the Schistosoma mansoni genome.

Precise, on-target CRISPR-Cas9 genome editing has been shown in Schistosoma mansoni, involving both non-homology end joining and homology-directed repair pathways. Here, we present a multiplexed CRISPR-Cas9 protocol for large transgene integration into the S. mansoni genome. We describe steps for deploying multiplexed ribonucleoprotein complexes (RNPs) and donor DNA preparation. We then detail procedures for introducing RNPs into schistosome eggs by square-wave electroporation in the presence of a 5' phosphorothioate-modified double-stranded donor transgene. For complete details on the use and execution of this protocol, please refer to Ittiprasert et al. (2023).1.

One-step knock-in of two antimicrobial peptide transgenes at multiple loci of catfish by CRISPR/Cas9-mediated multiplex genome engineering.

CRISPR/Cas9-mediated multiplex genome editing (MGE) conventionally uses multiple single-guide RNAs (sgRNAs) for gene-targeted mutagenesis via the non-homologous end joining (NHEJ) pathway. MGE has been proven to be highly efficient for functional gene disruption/knockout (KO) at multiple loci in mammalian cells or organisms. However, in the absence of a DNA donor, this approach is limited to small indels without transgene integration. Here, we establish the linear double-stranded DNA (dsDNA) and double-cut plasmid (dcPlasmid) combination-assisted MGE in channel catfish (Ictalurus punctatus), allowing combinational deletion mutagenesis and transgene knock-in (KI) at multiple sites through NHEJ/homology-directed repair (HDR) pathway in parallel. In this study, we used single-sgRNA-based genome editing (ssGE) and multi-sgRNA-based MGE (msMGE) to replace the luteinizing hormone (lh) and melanocortin-4 receptor (mc4r) genes with the cathelicidin (As-Cath) transgene and the myostatin (two target sites: mstn1, mstn2) gene with the cecropin (Cec) transgene, respectively. A total of 9000 embryos were microinjected from three families, and 1004 live fingerlings were generated and analyzed. There was no significant difference in hatchability (all P > 0.05) and fry survival (all P > 0.05) between ssGE and msMGE. Compared to ssGE, CRISPR/Cas9-mediated msMGE assisted by the mixture of dsDNA and dcPlasmid donors yielded a higher knock-in (KI) efficiency of As-Cath (19.93 %, [59/296] vs. 12.96 %, [45/347]; P = 0.018) and Cec (22.97 %, [68/296] vs. 10.80 %, [39/361]; P = 0.003) transgenes, respectively. The msMGE strategy can be used to generate transgenic fish carrying two transgenes at multiple loci. In addition, double and quadruple mutant individuals can be produced with high efficiency (36.3 % âˆ¼ 71.1 %) in one-step microinjection. In conclusion, we demonstrated that the CRISPR/Cas9-mediated msMGE allows the one-step generation of simultaneous insertion of the As-Cath and Cec transgenes at four sites, and the simultaneous disruption of the lh, mc4r, mstn1 and mstn2 alleles. This msMGE system, aided by the mixture donors, promises to pioneer a new dimension in the drive and selection of multiple designated traits in other non-model organisms.

Liver directed adeno-associated viral vectors to treat metabolic disease.

The liver is the metabolic center of the body and an ideal target for gene therapy of inherited metabolic disorders (IMDs). Adeno-associated viral (AAV) vectors can deliver transgenes to the liver with high efficiency and specificity and a favorable safety profile. Recombinant AAV vectors contain only the transgene cassette, and their payload is converted to non-integrating circular double-stranded DNA episomes, which can provide stable expression from months to years. Insights from cellular studies and preclinical animal models have provided valuable information about AAV capsid serotypes with a high liver tropism. These vectors have been applied successfully in the clinic, particularly in trials for hemophilia, resulting in the first approved liver-directed gene therapy. Lessons from ongoing clinical trials have identified key factors affecting efficacy and safety that were not readily apparent in animal models. Circumventing pre-existing neutralizing antibodies to the AAV capsid, and mitigating adaptive immune responses to transduced cells are critical to achieving therapeutic benefit. Combining the high efficiency of AAV delivery with genome editing is a promising path to achieve more precise control of gene expression. The primary safety concern for liver gene therapy with AAV continues to be the small risk of tumorigenesis from rare vector integrations. Hepatotoxicity is a key consideration in the safety of neuromuscular gene therapies which are applied at substantially higher doses. The current knowledge base and toolkit for AAV is well developed, and poised to correct some of the most severe IMDs with liver-directed gene therapy.

Site specific insertion of a transgene into the murine α-casein (CSN1S1) gene results in the predictable expression of a recombinant protein in milk.

Gene loci of highly expressed genes provide ideal sites for transgene expression. Casein genes are highly expressed in mammals leading to the synthesis of substantial amounts of casein proteins in milk. The α-casein (CSN1S1) gene has assessed as a site of transgene expression in transgenic mice and a mammary gland cell line. A transgene encoding an antibody light chain gene (A1L) was inserted into the α-casein gene using sequential homologous and site-specific recombination. Expression of the inserted transgene is directed by the α-casein promoter, is responsive to lactogenic hormone activation, leads to the synthesis of a chimeric α-casein/A1L transgene mRNA, and secretion of the recombinant A1L protein into milk. Transgene expression is highly consistent in all transgenic lines, but lower than that of the α-casein gene (4%). Recombinant A1L protein accounted for 0.5% and 1.6% of total milk protein in heterozygous and homozygous transgenic mice, respectively. The absence of the α-casein protein in homozygous A1L transgenic mice leads to a reduction of total milk protein and delayed growth of the pups nursed by these mice. Overall, the data demonstrate that the insertion of a transgene into a highly expressed endogenous gene is insufficient to guarantee its abundant expression.

Successful reversal of transgene silencing in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii has been successfully engineered to produce compounds of interest following transgene integration and heterologous protein expression. The advantages of this model include the availability of validated tools for bioengineering, its photosynthetic ability, and its potential use as biofuel. Despite this, breakthroughs have been hindered by its ability to silence transgene expression through epigenetic changes. Histone deacetylases (HDAC) are main players in gene expression. We hypothesized that transgene silencing can be reverted with chemical treatments using HDAC inhibitors. To analyze this, we transformed C. reinhardtii, integrating into its genome the mVenus reporter gene under the HSP70-rbcs2 promoter. From 384 transformed clones, 88 (22.9%) displayed mVenus positive (mVenus+ ) cells upon flow-cytometry analysis. Five clones with different fluorescence intensities were selected. The number of integrated copies was measured by qPCR. Transgene expression levels were followed over the growth cycle and upon SAHA treatment, using a microplate reader, flow cytometry, RT-qPCR, and western blot analysis. First, we observed that expression varies with the cell cycle, reaching a maximum level just before the stationary phase in all clones. Second, we uncovered that supplementation with HDAC inhibitors of the hydroxamate family, such as vorinostat (suberoylanilide-hydroxamic-acid, SAHA) at the initiation of culture increases the frequency (% of mVenus+ cells) and the level of transgene expression per cell over the whole growth cycle, through histone deacetylase inhibition. Thus, we propose a new tool to successfully trigger the expression of heterologous proteins in the green algae C. reinhardtii, overcoming its main obstacle as an expression platform.

RNA Aptamer-Mediated Gene Activation Systems for Inducible Transgene Expression in Animal Cells.

RNA expression analyses can be used to obtain various information from inside cells, such as physical conditions, the chemical environment, and endogenous signals. For detecting RNA, the system regulating intracellular gene expression has the potential for monitoring RNA expression levels in real time within living cells. Synthetic biology provides powerful tools for detecting and analyzing RNA inside cells. Here, we devised an RNA aptamer-mediated gene activation system, RAMGA, to induce RNA-triggered gene expression activation by employing an inducible complex formation strategy grounded in synthetic biology. This methodology connects DNA-binding domains and transactivators through target RNA using RNA-binding domains, including phage coat proteins. MS2 bacteriophage coat protein fused with a transcriptional activator and PP7 bacteriophage coat protein fused with the tetracycline repressor (tetR) can be bridged by target RNA encoding MS2 and PP7 stem-loops, resulting in transcriptional activation. We generated recombinant CHO cells containing an inducible GFP expression module governed by a minimal promoter with a tetR-responsive element. Cells carrying the trigger RNA exhibited robust reporter gene expression, whereas cells lacking it exhibited no expression. GFP expression was upregulated over 200-fold compared with that in cells without a target RNA expression vector. Moreover, this system can detect the expression of mRNA tagged with aptamer tags and modulate reporter gene expression based on the target mRNA level without affecting the expression of the original mRNA-encoding gene. The RNA-triggered gene expression systems developed in this study have potential as a new platform for establishing gene circuits, evaluating endogenous gene expression, and developing novel RNA detectors.

Interspecies Scaling of Transgene Products for Viral Vector Gene Therapies: Method Assessment Using Data from Eleven Viral Vectors.

The prediction of transgene product expression in human is important to guide first-in-human (FIH) dose selection for viral vector-based gene replacement therapies. Recently, allometric scaling from preclinical data and interspecies normalization of dose-response (D-R) relationship have been used to predict human transgene product expression of adeno-associated virus (AAV) vectors. In this study, we assessed two interspecies allometric scaling methods and two dose-response methods in predicting human transgene product expression of nine intravenously administered AAV vectors, one intramuscularly administered AAV vector, and one intravesical administered adenoviral vector. Among the four methods, normalized D-R method generated the highest prediction accuracy, with geometric mean fold error (GMFE) of 2.9 folds and 75% predictions within fivefold deviations of observed human transgene product levels. The vg/kg-based D-R method worked well for locally delivered vectors but substantially overpredicted human transgene product levels of some hemophilia A and B vectors. For both intravenously and locally administered vectors, the prediction accuracy of allometric scaling using body weight^-0.25 (AS by W^-0.25) was superior to allometric scaling using log(body weight) (AS by logW). This study successfully extended the use of allometric scaling and interspecies D-R normalization methods for human transgene product prediction from intravenous viral vectors to locally delivered viral vectors.

Design and testing of a humanized porcine donor for xenotransplantation.

Recent human decedent model studies1,2 and compassionate xenograft use3 have explored the promise of porcine organs for human transplantation. To proceed to human studies, a clinically ready porcine donor must be engineered and its xenograft successfully tested in nonhuman primates. Here we describe the design, creation and long-term life-supporting function of kidney grafts from a genetically engineered porcine donor transplanted into a cynomolgus monkey model. The porcine donor was engineered to carry 69 genomic edits, eliminating glycan antigens, overexpressing human transgenes and inactivating porcine endogenous retroviruses. In vitro functional analyses showed that the edited kidney endothelial cells modulated inflammation to an extent that was indistinguishable from that of human endothelial cells, suggesting that these edited cells acquired a high level of human immune compatibility. When transplanted into cynomolgus monkeys, the kidneys with three glycan antigen knockouts alone experienced poor graft survival, whereas those with glycan antigen knockouts and human transgene expression demonstrated significantly longer survival time, suggesting the benefit of human transgene expression in vivo. These results show that preclinical studies of renal xenotransplantation could be successfully conducted in nonhuman primates and bring us closer to clinical trials of genetically engineered porcine renal grafts.

Death after High-Dose rAAV9 Gene Therapy in a Patient with Duchenne's Muscular Dystrophy.

We treated a 27-year-old patient with Duchenne's muscular dystrophy (DMD) with recombinant adeno-associated virus (rAAV) serotype 9 containing dSaCas9 (i.e., "dead" Staphylococcus aureus Cas9, in which the Cas9 nuclease activity has been inactivated) fused to VP64; this transgene was designed to up-regulate cortical dystrophin as a custom CRISPR-transactivator therapy. The dose of rAAV used was 1×1014 vector genomes per kilogram of body weight. Mild cardiac dysfunction and pericardial effusion developed, followed by acute respiratory distress syndrome (ARDS) and cardiac arrest 6 days after transgene treatment; the patient died 2 days later. A postmortem examination showed severe diffuse alveolar damage. Expression of transgene in the liver was minimal, and there was no evidence of AAV serotype 9 antibodies or effector T-cell reactivity in the organs. These findings indicate that an innate immune reaction caused ARDS in a patient with advanced DMD treated with high-dose rAAV gene therapy. (Funded by Cure Rare Disease.).

Spatial atlas of the mouse central nervous system at molecular resolution.

Spatially charting molecular cell types at single-cell resolution across the 3D volume is critical for illustrating the molecular basis of brain anatomy and functions. Single-cell RNA sequencing has profiled molecular cell types in the mouse brain1,2, but cannot capture their spatial organization. Here we used an in situ sequencing method, STARmap PLUS3,4, to profile 1,022 genes in 3D at a voxel size of 194 × 194 × 345 nm3, mapping 1.09 million high-quality cells across the adult mouse brain and spinal cord. We developed computational pipelines to segment, cluster and annotate 230 molecular cell types by single-cell gene expression and 106 molecular tissue regions by spatial niche gene expression. Joint analysis of molecular cell types and molecular tissue regions enabled a systematic molecular spatial cell-type nomenclature and identification of tissue architectures that were undefined in established brain anatomy. To create a transcriptome-wide spatial atlas, we integrated STARmap PLUS measurements with a published single-cell RNA-sequencing atlas1, imputing single-cell expression profiles of 11,844 genes. Finally, we delineated viral tropisms of a brain-wide transgene delivery tool, AAV-PHP.eB5,6. Together, this annotated dataset provides a single-cell resource that integrates the molecular spatial atlas, brain anatomy and the accessibility to genetic manipulation of the mammalian central nervous system.

Transgenic ferret models define pulmonary ionocyte diversity and function.

Speciation leads to adaptive changes in organ cellular physiology and creates challenges for studying rare cell-type functions that diverge between humans and mice. Rare cystic fibrosis transmembrane conductance regulator (CFTR)-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans1,2, but limited presence and divergent biology in the proximal trachea of mice has prevented the use of traditional transgenic models to elucidate ionocyte functions in the airway. Here we describe the creation and use of conditional genetic ferret models to dissect pulmonary ionocyte biology and function by enabling ionocyte lineage tracing (FOXI1-CreERT2::ROSA-TG), ionocyte ablation (FOXI1-KO) and ionocyte-specific deletion of CFTR (FOXI1-CreERT2::CFTRL/L). By comparing these models with cystic fibrosis ferrets3,4, we demonstrate that ionocytes control airway surface liquid absorption, secretion, pH and mucus viscosity-leading to reduced airway surface liquid volume and impaired mucociliary clearance in cystic fibrosis, FOXI1-KO and FOXI1-CreERT2::CFTRL/L ferrets. These processes are regulated by CFTR-dependent ionocyte transport of Cl- and HCO3-. Single-cell transcriptomics and in vivo lineage tracing revealed three subtypes of pulmonary ionocytes and a FOXI1-lineage common rare cell progenitor for ionocytes, tuft cells and neuroendocrine cells during airway development. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of cystic fibrosis airway disease. These studies provide a road map for using conditional genetics in the first non-rodent mammal to address gene function, cell biology and disease processes that have greater evolutionary conservation between humans and ferrets.

Evidence of a novel silencing effect on transgenes in the Arabidopsis thaliana sperm cell.

We encountered unexpected transgene silencing in Arabidopsis thaliana sperm cells; transgenes encoding proteins with no specific intracellular localization (cytoplasmic proteins) were silenced transcriptionally or posttranscriptionally. The mRNA of cytoplasmic protein transgenes tagged with a fluorescent protein gene was significantly reduced, resulting in undetectable fluorescent protein signals in the sperm cell. Silencing of the cytoplasmic protein transgenes in the sperm cell did not affect the expression of either its endogenous homologous genes or cotransformed transgenes encoding a protein with targeted intracellular localization. This transgene silencing in the sperm cell persisted in mutants of the major gene silencing machinery including DNA methylation. The incomprehensible, yet real, transgene silencing phenotypes occurring in the sperm cell could mislead the interpretation of experimental results in plant reproduction, and this Commentary calls attention to that risk and highlights details of this novel cytoplasmic protein transgene silencing.

Increased hyaluronan by naked mole-rat Has2 improves healthspan in mice.

Abundant high-molecular-mass hyaluronic acid (HMM-HA) contributes to cancer resistance and possibly to the longevity of the longest-lived rodent-the naked mole-rat1,2. To study whether the benefits of HMM-HA could be transferred to other animal species, we generated a transgenic mouse overexpressing naked mole-rat hyaluronic acid synthase 2 gene (nmrHas2). nmrHas2 mice showed an increase in hyaluronan levels in several tissues, and a lower incidence of spontaneous and induced cancer, extended lifespan and improved healthspan. The transcriptome signature of nmrHas2 mice shifted towards that of longer-lived species. The most notable change observed in nmrHas2 mice was attenuated inflammation across multiple tissues. HMM-HA reduced inflammation through several pathways, including a direct immunoregulatory effect on immune cells, protection from oxidative stress and improved gut barrier function during ageing. These beneficial effects were conferred by HMM-HA and were not specific to the nmrHas2 gene. These findings demonstrate that the longevity mechanism that evolved in the naked mole-rat can be exported to other species, and open new paths for using HMM-HA to improve lifespan and healthspan.

Targeted insertion and reporter transgene activity at a gene safe harbor of the human blood fluke, Schistosoma mansoni.

The identification and characterization of genomic safe harbor sites (GSHs) can facilitate consistent transgene activity with minimal disruption to the host cell genome. We combined computational genome annotation and chromatin structure analysis to predict the location of four GSHs in the human blood fluke, Schistosoma mansoni, a major infectious pathogen of the tropics. A transgene was introduced via CRISPR-Cas-assisted homology-directed repair into one of the GSHs in the egg of the parasite. Gene editing efficiencies of 24% and transgene-encoded fluorescence of 75% of gene-edited schistosome eggs were observed. The approach advances functional genomics for schistosomes by providing a tractable path for generating transgenics using homology-directed, repair-catalyzed transgene insertion. We also suggest that this work will serve as a roadmap for the development of similar approaches in helminths more broadly.

Combination of MAR and intron increase transgene expression of episomal vectors in CHO cells.

Previous work has shown that the EF-1α promoter of episomal vectors maintains high-level transgene expression in stably transfected Chinese hamster ovary (CHO) cells. However, the transgene expression levels need to be further increased. Here, we first incorporated matrix attachment regions (MARs), ubiquitous chromatin opening element (UCOE), stabilizing anti repressor elements 40 (STAR 40) elements into episomal vector at different sites and orientations, and systemically assessed their effects on transgene expression in transfected CHO-K1 cells. Results showed that enhanced green fluorescent protein (eGFP) expression levels increased remarkably when MAR X-29 was inserted upstream of the promoter, followed by the insertion of MAR1 downstream of the poly A, and the orientation had no significant effect. Moreover, MAR X-29 combined with human cytomegalovirus intron (hCMVI) yielded the highest transgene expression levels (4.52-fold). Transgene expression levels were not exclusively dependent on transgene copy numbers and were not related to the mRNA expression level. In addition, vector with MAR X-29+hCMVI can induce herpes simplex virus thymidine kinase (HSV-TK) protein expression, and the HSV-TK protein showed a cell-killing effect and an obvious bystander effect on HCT116 cells. In conclusion, the combination of MAR X-29 and hCMV intron can achieve high efficiency transgene expression mediated by episomal vectors in CHO-K1 cells.

High-throughput library transgenesis in Caenorhabditis elegans via Transgenic Arrays Resulting in Diversity of Integrated Sequences (TARDIS).

High-throughput transgenesis using synthetic DNA libraries is a powerful method for systematically exploring genetic function. Diverse synthesized libraries have been used for protein engineering, identification of protein-protein interactions, characterization of promoter libraries, developmental and evolutionary lineage tracking, and various other exploratory assays. However, the need for library transgenesis has effectively restricted these approaches to single-cell models. Here, we present Transgenic Arrays Resulting in Diversity of Integrated Sequences (TARDIS), a simple yet powerful approach to large-scale transgenesis that overcomes typical limitations encountered in multicellular systems. TARDIS splits the transgenesis process into a two-step process: creation of individuals carrying experimentally introduced sequence libraries, followed by inducible extraction and integration of individual sequences/library components from the larger library cassette into engineered genomic sites. Thus, transformation of a single individual, followed by lineage expansion and functional transgenesis, gives rise to thousands of genetically unique transgenic individuals. We demonstrate the power of this system using engineered, split selectable TARDIS sites in Caenorhabditis elegans to generate (1) a large set of individually barcoded lineages and (2) transcriptional reporter lines from predefined promoter libraries. We find that this approach increases transformation yields up to approximately 1000-fold over current single-step methods. While we demonstrate the utility of TARDIS using C. elegans, in principle the process is adaptable to any system where experimentally generated genomic loci landing pads and diverse, heritable DNA elements can be generated.

Transgenesis ­ the ability to insert foreign genetic material (known as transgenes) in to the genome of an organism ­ has revolutionized biological research. This approach has made it possible for scientists to study the role of specific genes and to produce animal models which mimic aspects of human diseases. For transgenes to be maintained and passed down to future generations, they must be introduced into germ cells which will go on to form the egg and sperm of the organism. However, despite advances in genetic engineering, this process (called 'specific transgenesis') is still laborious and time-consuming, and limits researchers to working with only a small number of known DNA sequences at a time. In contrast, 'exploratory transgenesis' ­ where dozens of transgenes from a library of DNA sequences are introduced simultaneously into multiple individuals ­ is more efficient and allows for more large-scale experiments. However, this approach can only be done with single-celled organisms like bacteria, and remains virtually impossible in laboratory animals like worms or mice. Stevenson et al. therefore set out to boost the efficiency of exploratory transgenesis in a commonly used laboratory animal, the roundworm Caenorhabditis elegans. To do this, they used the 'library' principle of exploratory transgenesis in order to develop a new resource called TARDIS (short for, Transgenic Arrays Resulting in Diversity of Integrated Sequences). First, Stevenson et al. genetically engineered worms to carry a 'landing site' for foreign DNA. Next, a library of transgenes and a mechanism which cuts pieces of DNA and pastes them into the landing site were introduced into the germ cells of these worms using traditional methods. The worms were then bred to generate a large population of offspring that had inherited this array of foreign DNA sequences. Finally, the 'cut and paste' mechanism was switched on and a random transgene was inserted into the landing site in the genome. This resulted in thousands of worms which each had a unique genetic modification that can be passed on to future generations. These results show for the first time that larger-scale transgenesis experiments are possible in multi-cellular animals. In the future, Stevenson et al. hope that TARDIS can be adapted to different organisms and allow researchers to carry out experiments that were not previously possible.

Plastid dsRNA transgenes trigger phased small RNA-based gene silencing of nuclear-encoded genes.

Plastid transformation technology has been widely used to express traits of potential commercial importance, though the technology has been limited to traits that function while sequestered in the organelle. Prior research indicates that plastid contents can escape from the organelle, suggesting a possible mechanism for engineering plastid transgenes to function in other cellular locations. To test this hypothesis, we created tobacco (Nicotiana tabacum cv. Petit Havana) plastid transformants that express a fragment of the nuclear-encoded Phytoene desaturase (PDS) gene capable of catalyzing post-transcriptional gene silencing if RNA escapes into the cytoplasm. We found multiple lines of direct evidence that plastid-encoded PDS transgenes affect nuclear PDS gene silencing: knockdown of the nuclear-encoded PDS mRNA and/or its apparent translational inhibition, biogenesis of 21-nucleotide (nt) phased small interfering RNAs (phasiRNAs), and pigment-deficient plants. Furthermore, plastid-expressed dsRNA with no cognate nuclear-encoded pairing partner also produced abundant 21-nt phasiRNAs in the cytoplasm, demonstrating that a nuclear-encoded template is not required for siRNA biogenesis. Our results indicate that RNA escape from plastids to the cytoplasm occurs generally, with functional consequences that include entry into the gene silencing pathway. Furthermore, we uncover a method to produce plastid-encoded traits with functions outside of the organelle and open additional fields of study in plastid development, compartmentalization, and small RNA biogenesis.

The impact of expression vector position on transgene transcription allows for rational expression vector design in a targeted integration system.

Site-specific integration (SSI) technology has emerged as an effective approach by the pharmaceutical industry for the development of recombinant Chinese hamster ovary (CHO) cell lines. While SSI systems have been demonstrated to be effective for the development of CHO cell lines, they can be limiting in terms of both transgene expression and in the case of multi-specifics, the ability to generate the correct product of interest. To maximize the performance of Pfizer's dual SSI expression system for expressing monoclonal and multi-specific antibodies, we used a novel approach to investigate the positional effect of transgenes within expression vectors by engineering nucleotide polymorphisms (NP)s to use as biomarkers to track the level of transcript output from each expression vector position. We observed differences in transcript level for two different transgenes across all four expression vector positions interrogated. We then applied these learnings to rationally design expression vectors for six different mAbs and a multi-specific antibody. We showed enhanced productivity and optimal product quality when compared to a conventional expression vector topology. The learnings gained here can potentially aid in the determination of optimal vector topologies for several IgG-like multi-specific formats.

Stable expression of large transgenes via the knock-in of an integrase-deficient lentivirus.

The targeted insertion and stable expression of a large genetic payload in primary human cells demands methods that are robust, efficient and easy to implement. Large payload insertion via retroviruses is typically semi-random and hindered by transgene silencing. Leveraging homology-directed repair to place payloads under the control of endogenous essential genes can overcome silencing but often results in low knock-in efficiencies and cytotoxicity. Here we report a method for the knock-in and stable expression of a large payload and for the simultaneous knock-in of two genes at two endogenous loci. The method, which we named CLIP (for 'CRISPR for long-fragment integration via pseudovirus'), leverages an integrase-deficient lentivirus encoding a payload flanked by homology arms and 'cut sites' to insert the payload upstream and in-frame of an endogenous essential gene, followed by the delivery of a CRISPR-associated ribonucleoprotein complex via electroporation. We show that CLIP enables the efficient insertion and stable expression of large payloads and of two difficult-to-express viral antigens in primary T cells at low cytotoxicity. CLIP offers a scalable and efficient method for manufacturing engineered primary cells.