Ongoing development of recombinant vectors based on adeno-associated virus (rAAV) is providing an increasingly powerful and widely used toolkit for gene transfer and genome editing applications. While conceptually simple, the system harbors considerable complexity that presents many potential pitfalls for the inexperienced user. The short inverted terminal repeats (ITRs) can prove to be particularly problematic during vector engineering due to inherent instability necessitating diligent quality control measures during vector manufacture. This is especially important from a clinical standpoint when consistent purity and potency are paramount, and all components of the system are rigorously scrutinized by regulatory agencies. Despite the discovery over 30 years ago that the AAV ITRs are the only cis-acting elements of the virus required for vector production, there is a scarcity of reviews specifically focused on these complex elements. This review provides an overview of the ITR with the dual purpose of acting as a user's guide in the application of AAV vector technology and as a roadmap for ongoing vector development and optimization.

Adeno-associated viral (AAV) vectors have shown great promise in gene delivery as evidenced by recent FDA approvals. Despite efforts to optimize manufacturing for good manufacturing practice (GMP) productions, few academic laboratories have the resources to assess vector composition. One critical component of vector quality is packaged genome fidelity. Errors in viral genome replication and packaging can result in the incorporation of faulty genomes with mutations, truncations, or rearrangements, compromising vector potency. Thus, sequence validation of packaged genome composition is an important quality control (QC), even in academic settings. We developed Fast-Seq, an end-to-end method for extraction, purification, sequencing, and data analysis of packaged single-stranded AAV (ssAAV) genomes intended for non-GMP preclinical environments. We validated Fast-Seq on ssAAV vectors with three different genome compositions (CAG-GFP, CAG-tdTomato, EF1α-FLuc), three different genome sizes (2.9, 3.6, 4.4 kb), packaged in four different capsid serotypes (AAV1, AAV2, AAV5, and AAV8), and produced using the two most common production methods (Baculovirus-Sf9 and human HEK293), from both common commercial vendors and academic core facilities supplying academic laboratories. We achieved an average genome coverage of >1,400 × and an average inverted terminal repeat coverage of >280 × , despite the many differences in composition of each ssAAV sample. When compared with other ssAAV next-generation sequencing (NGS) methods for GMP settings, Fast-Seq has several unique advantages: Tn5 transposase-based fragmentation rather than sonication, 125 × less input DNA, simpler adapter ligation, compatibility with commonly available inexpensive sequencing instruments, and free open-source data analysis code in a preassembled customizable Docker container designed for novices. Fast-Seq can be completed in 18 h, is more cost-effective than other NGS methods, and is more accurate than Sanger sequencing, which is generally only applied at 1–2 × sequencing depth. Fast-Seq is a rapid, simple, and inexpensive methodology to validate packaged ssAAV genomes in academic settings.

The detailed characterization of biological nanoparticles is of paramount importance for various industrial sectors, as for production of viral therapeutics. More recently, technologies that allow real-time quantification with simultaneous sizing and determination of surface potentials of virus particles in solution have been developed. In this study, nanoparticle tracking analysis (NTA) was applied to determine the size and the zeta potential of human adenovirus type 5 (AdV5), one the most frequently used therapeutic/oncolytic agents and viral vectors. Virus aggregation was detected, and the kinetics of the dissolution of virus aggregates were studied in real time. In addition, advanced fluorescence detection of AdV5 was performed enabling the measurements in matrices and discrimination of viral subpopulations. It was shown that NTA is an efficient approach for investigating infectious viruses in a live viewing mode. Consequently, NTA provides a promising methodology for virus particle detection and analysis in real time beyond assays requiring nucleic acids or infectivity.

Viral vectors are complex drugs that pose a particular challenge for manufacturing. Previous studies have shown that, unlike small-molecule drugs, vector preparations do not yield a collection of identical particles. Instead, a mixture of particles that vary in capsid stoichiometry and impurities is created, which may differ from lot to lot. The consequences of this are unclear, but conflicting reports regarding the biological properties of vectors, including transduction patterns, suggest that this variability may have an effect. However, other variables, including differences in animal strains and techniques, make it difficult to identify a cause. Here, we report lot-to-lot variation in spinal cord gray matter transduction following intrathecal delivery of self-complementary adeno-associated virus serotype 9 vectors. Eleven lots of vector were evaluated from six vector cores, including one preclinical/Good Laboratory Practice lot. Eight of the lots, including the preclinical lot, failed to transduce the gray matter, whereas the other three provided robust transduction. The cause for this variation is unknown, but it did not correlate with vector titer, buffer, or purification method. These results highlight the need to identify the cause of this variation and to develop improved production and quality control methods to ensure lot-to-lot consistency of vector potency.

Non-small cell lung cancer (NSCLC) denotes the commonest type of lung cancers with high mortality globally. Long non-coding RNAs (lncRNAs) with differential expression have been indicated to be participants in the pathogenesis and development of cancer. However, the precise role of lncRNAs in NSCLC is still largely obscure. In this study, we explored a newly-discovered intergenic lncRNA LINC00958 in NSCLC. First of all, the online databases suggested that LINC00958 was low expressed in human normal lung tissues but upregulated in LUSC tissues. Besides, the upregulation of LINC00958 in both LUAD and LUSC cell lines was easily found when comparing to the normal BEAS-2B cells. In addition, we elucidated that knockdown of LINC00958 led to impaired proliferation, induced apoptosis and hampered migration in LUAD cells. Moreover, a typical oncogenic pathway, JNK signaling, was verified to be involved in LINC00958-contributed LUAD development. Of note, we explained that LINC00958 exerted the tumor-promoting function in LUAD by enhancing the transactivation of p-c-JUN through activating JNK signaling. Meanwhile, we also revealed that LINC00958 was transcriptionally regulated by c-JUN. Additionally, above findings were also suitable for LUSC cells. By and large, our work illustrated that LINC00958 facilitates tumorigenesis in NSCLC by activating JNK/c-JUN signaling pathway, indicating a new road for diagnosis and treatment of both LUAD and LUSC.

Adeno-associated viral (AAV) vectors have shown great promise in gene delivery as evidenced by recent FDA approvals. Despite efforts to optimize manufacturing for GMP productions, few academic labs have the resources to assess vector composition. One critical component of vector quality is packaged genome fidelity. Errors in viral genome replication and packaging can result in the incorporation of faulty genomes with mutations, truncations or rearrangements, compromising vector potency. Thus, sequence validation of packaged genome composition is an important quality control, even in academic settings. We developed Fast-Seq, an end-to-end method for extraction, purification, sequencing and data analysis of packaged ssAAV genomes intended for non-GMP preclinical environments. We validated Fast-Seq on ssAAV vectors with 3 different genome compositions (CAG-tdTomato, EF1⍺-FLuc, CAG-GFP), 3 different genome sizes (2.9kb, 3.6kb, 4.3kb), packaged in 4 different capsid serotypes (AAV1, AAV2, AAV5, AAV8), and produced using the two most common production methods (Baculovirus-Sf9, human HEK293), from both common commercial vendors and academic core facilities supplying academic laboratories. We achieved an average genome coverage of >1,400X and an average ITR coverage of >280X, despite the many differences in composition of each ssAAV sample. When compared to other ssAAV NGS methods for GMP settings, Fast-Seq has several unique advantages: Tn5 transposase-based fragmentation rather than sonication, 125x less input DNA, simpler adapter ligation, compatibility with commonly available inexpensive sequencing instruments, and free open-source data analysis code in a pre-assembled customizable Docker container designed for novices. Fast-Seq can be completed in 18 hours, is more cost-effective than other NGS methods, and is more accurate than Sanger sequencing which is generally only applied at 1-2x sequencing depth. Fast-Seq is a rapid, simple, and inexpensive methodology to validate packaged ssAAV genomes in academic settings.

Non-small cell lung cancer (NSCLC) denotes the most common type of lung cancers with high mortality globally. Long non-coding RNAs (lncRNAs) with differential expression have been indicated to be participants in the pathogenesis and development of cancer. However, the precise role of lncRNAs in NSCLC is still largely obscure. In this study, we explored a newly discovered intergenic lncRNA LINC00958 in NSCLC. First of all, the online databases suggested that LINC00958 was slightly expressed in human normal lung tissues but upregulated in LUSC tissues. Besides, the upregulation of LINC00958 in both lung adenocarcinoma (LUAD) and LUSC cell lines was easily found when compared with the normal BEAS-2B cells. In addition, we elucidated that knockdown of LINC00958 led to impaired proliferation, induced apoptosis, and hampered migration in LUAD cells. Moreover, a typical oncogenic pathway, JNK signaling, was verified to be involved in LINC00958-contributed LUAD development. Of note, we explained that LINC00958 exerted the tumor-promoting function in LUAD by enhancing the transactivation of p-c-JUN through activating JNK signaling. Meanwhile, we also revealed that LINC00958 was transcriptionally regulated by c-JUN. In addition, earlier findings were also suitable for LUSC cells. By and large, our work illustrated that LINC00958 facilitates tumorigenesis in NSCLC by activating the JNK/c-JUN signaling pathway, indicating a new road for diagnosis and treatment of both LUAD and LUSC.

Cerebral infarction is a leading cause of death, which calls for effective prevention and treatment. Transplant of neural stem cells (NSCs) is a potential therapeutic treatment to cerebral infarction although its efficacy still needs to be improved. Overexpression of Hypoxia-inducible factor 1α (HIF-1α) has been shown to enhance the protective effects of stem cell transplant on cerebral infarction. The expression of HIF-1α is predicted to be regulated by miR-155-5p. Therefore, we regulated the expression of miR-155-5p in NSCs and evaluated the effects of miR-155-5p-regulated NSC transplant on cerebral infarction. We inhibited miR-155-5p expression in NSCs by overexpressing miR-155-5p inhibitor. HIF-1α expression, cell viability and the expression of apoptosis markers were examined. We established the middle cerebral artery occlusion (MCAO) rat model, and the infarct volume, neurobehavioral outcomes, inflammation and oxidative stress were evaluated after NSC transplant. miR-155-5p directly targeted HIF-1α and negatively regulated its expression. Inhibition of miR-155-5p enhanced cell viability and prevented cell apoptosis. Transplant of miR-155-5p-inhibited NSCs significantly decreased infarct volume, improved neurobehavioral outcomes of MCAO rats. Transplant of miR-155-5p-inhibited NSCs significantly inhibited inflammation and oxidative stress. Inhibition of miR-155-5p in NSCs resulted in enhanced protection against cerebral infarction after NSC transplant.

Recombinant adeno-associated viruses (rAAVs) are excellent vectors for gene delivery. However, current Sf9/Cap-Rep packaging cell line-dependent OneBac systems still lack versatility and flexibility for large-scale production of rAAVs. In this study, we developed an improved OneBac system that includes a novel dual-function baculovirus expression vector (BEV) termed BEV/Cap-(ITR-GOI) that carries both the AAV Cap gene and rAAV genome inverted terminal repeat (ITR) sequences flanking the gene of interest (GOI), a versatile Sf9-GFP/Rep packaging cell line that harbors silent copies of the AAV2 Rep gene that can be expressed after BEV infection, and constitutively expressed green fluorescent protein (GFP) reporter genes to facilitate cell line screening. The BEV/Cap-(ITR-GOI) construct allows flexibility to switch among different Cap gene serotypes using simple BEV reconstruction, and is stable for at least five serial passages. Furthermore, the Sf9-GFP/Rep stable cell line is versatile for production of different rAAV serotypes. The yield levels for rAAV2, rAAV8, and rAAV9 exceeded 105 vector genomes (VG) per cell, which is similar to other currently available large-scale rAAV production systems. The new Bac system-derived rAAVs have biophysical properties similar to HEK293 cell-derived rAAVs, as well as high quality and activity. In summary, the novel Sf9-GFP/Rep packaging cell line-dependent OneBac system can facilitate large-scale rAAV production and rAAV-based gene therapy.

Cerebral infarction is a leading cause of death, which calls for effective prevention and treatment. Transplant of neural stem cells (NSCs) is a potential therapeutic treatment to cerebral infarction although its efficacy still needs to be improved. Overexpression of hypoxia-inducible factor 1α (HIF-1α) has been shown to enhance the protective effects of stem cell transplant on cerebral infarction. The expression of HIF-1α is predicted to be regulated by miR-155-5p. Therefore, we regulated the expression of miR-155-5p in NSCs and evaluated the effects of miR-155-5p-regulated NSC transplant on cerebral infarction. We inhibited miR-155-5p expression in NSCs by overexpressing miR-155-5p inhibitor. HIF-1α expression, cell viability, and the expression of apoptosis markers were examined. We established the middle cerebral artery occlusion (MCAO) rat model, and the infarct volume, neurobehavioral outcomes, inflammation, and oxidative stress were evaluated after NSC transplant. miR-155-5p directly targeted HIF-1α and negatively regulated its expression. Inhibition of miR-155-5p enhanced cell viability and prevented cell apoptosis. Transplant of miR-155-5p-inhibited NSCs significantly decreased infarct volume and improved neurobehavioral outcomes of MCAO rats. Transplant of miR-155-5p-inhibited NSCs significantly inhibited inflammation and oxidative stress. Inhibition of miR-155-5p in NSCs resulted in enhanced protection against cerebral infarction after NSC transplant.