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PubMed2017

Future of rAAV Gene Therapy: Platform for RNAi, Gene Editing, and Beyond.

The use of recombinant adeno-associated viruses (rAAVs) ushered in a new millennium of gene transfer for therapeutic treatment of a number of conditions, including congenital blindness, hemophilia, and spinal muscular atrophy. rAAV vectors have remarkable staying power from a therapeutic standpoint, withstanding several ebbs and flows. As new technologies such as clustered regularly interspaced short palindromic repeat genome editing emerge, it is now the delivery tool-the AAV vector-that is the stalwart. The long-standing safety of this vector in a multitude of clinical settings makes rAAV a selling point in the advancement of approaches for gene replacement, gene knockdown, gene editing, and genome modification/engineering. The research community is building on these advances to develop more tailored delivery approaches and to tweak the genome in new and unique ways. Intertwining these approaches with newly engineered rAAV vectors is greatly expanding the available tools to manipulate gene expression with a therapeutic intent.

Dependovirus↗
PubMed1991

Construction and use of halobacterial shuttle vectors and further studies on Haloferax DNA gyrase.

We report here on advances made in the construction of plasmid shuttle vectors suitable for genetic manipulations in both Escherichia coli and halobacteria. Starting with a 20.4-kb construct, pMDS1, new vectors were engineered which were considerably smaller yet retained several alternative cloning sites. A restriction barrier observed when plasmid DNA was transferred into Haloferax volcanii cells was found to operate via adenine methylation, resulting in a 10(3) drop in transformation efficiency and the loss of most constructs by incorporation of the resistance marker into the chromosome. Passing shuttle vectors through E. coli dam mutants effectively avoided this barrier. Deletion analysis revealed that the gene(s) for autonomous replication of pHK2 (the plasmid endogenous to Haloferax strain Aa2.2 and used in the construction of pMDS1) was located within a 4.2-kb SmaI-KpnI fragment. Convenient restriction sites were identified near the termini of the novobiocin resistance determinant (gyrB), allowing the removal of flanking sequences (including gyrA). These deletions did not appear to significantly affect transformation efficiencies or the novobiocin resistance phenotype of halobacterial transformants. Northern blot hybridization with strand- and gene-specific probes identified a single gyrB-gyrA transcript of 4.7 kb. This is the first demonstration in prokaryotes that the two subunits of DNA gyrase may be cotranscribed.

Blotting, Northern↗
PubMed1988

Fowlpox virus as a vector in non-avian species.

Examination of the members of the Poxvirus family reveals a large and diverse group with members infecting almost every animal species. To a large extent, members of the individual genera have a broad host range and infect a number of animal species, although, as Baxby has pointed out, successful experimental inoculation of a species does not necessarily mean that this species provides a natural host for that virus. Avipox viruses, together with swinepox virus occupy a somewhat unique position in possessing a restricted host range. We have taken advantage of this restricted host range in fowlpox virus to engineer recombinant vector viruses for use in the poultry industry and in vaccination of non-avian species.

Animals↗
PubMed1992

New plasmid vector and host system for genetic engineering in Bacillus sphaericus.

A new plasmid, pNQ116, was constructed in Bacillus sphaericus by cloning a promoter fragment from B. sphaericus Ts-1 into pNQ112. The plasmid (CmrKmr, 5.23 kb) contains a restriction endonuclease polylinker used for cloning foreign genes, and its cat-86 gene is expressed at high levels from the Ts-1 promoter. This plasmid vector has been transformed into B. sphaericus AS 1.270, AS 1.465, AS 1.469, and 2362, at frequencies of 10(2)-10(3) transformants per microgram of DNA, and is maintained stably under nonselective conditions in these host strains. The presence of pNQ116 in B. sphaericus 2362 does ot interfere with the mosquito larvicidal activity of the organism.

Animals↗
PubMed1989

Epidermal growth factor binding induces a conformational change in the external domain of its receptor.

To study the properties of the extracellular epidermal growth factor (EGF) binding domain of the human EGF receptor, we have infected insect cells with a suitably engineered baculovirus vector containing the cDNA encoding the entire ectodomain of the parent molecule. This resulted in a correctly folded, stable, 110 kd protein which possessed an EGF binding affinity of 200 nM. The protein was routinely purified in milligram amounts from 1 litre insect cell cultures using a series of three standard chromatographic steps. The properties of the ectodomain were studied before and after the addition of different EGF ligands, using both circular dichroism and fluorescence spectroscopic techniques. A secondary structural analysis of the far UV CD spectrum of the ectodomain indicated significant proportions of alpha-helix and beta-sheet in agreement with a published model of the EGF receptor. The ligand additions to the receptor showed differences in both the near- and far-UV CD spectra, and were similar for each ligand used, suggesting similar conformational differences between uncomplexed and complexed receptor. Steady-state fluorescence measurements indicated that the tryptophan residues present in the ectodomain are buried and that the solvent-accessible tryptophans in the ligands become buried on binding the receptor. The rotational correlation times measured by fluorescence anisotropy decay for the receptor-ligand complexes were decreased from 6 to 2.5 ns in each case. This may indicate a perturbation of the tryptophan environment of the receptor on ligand binding. Ultracentrifugation studies showed that no aggregation occurred on ligand addition, so this could not explain the observed differences from CD or fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Human preproendothelin-1 is converted into active endothelin-1 by baculovirus-infected insect cells.

To investigate biochemical and biological parameters involved in preproendothelin-1 (preproET-1) maturation we infected Spodoptera frugiperda (Sf21) cells with a suitable engineered baculovirus vector carrying the cDNA encoding the entire human 212 amino acids precursor. Culture supernatants were tested by RIA using an anti-ET-1 serum, ET-1-like immunoreactive material (IRM) was detected in the infected Sf21 cells but not in control, wild-type or mock-infected cells. Fractionation of the culture supernatant by RP-HPLC coupled to an ET-1 specific RIA yielded two main peaks corresponding to the retention times of human bigET-1 and ET-1. Furthermore, culture supernatant of preproET-1 expressing Sf21 cells elicited a characteristic dose-response vasoconstrictive activity on rabbit vena cava, consistent with the amount of ET-1 as estimated by RP-HPLC coupled to RIA. These results suggest that insect cells possess the enzymatic activities necessary for human preproET-1 full maturation even though no such peptide has ever been found in insect cells.

Animals↗
PubMed2009

A myocardium tropic adeno-associated virus (AAV) evolved by DNA shuffling and in vivo selection.

To engineer gene vectors that target striated muscles after systemic delivery, we constructed a random library of adeno-associated virus (AAV) by shuffling the capsid genes of AAV serotypes 1 to 9, and screened for muscle-targeting capsids by direct in vivo panning after tail vein injection in mice. After 2 rounds of in vivo selection, a capsid gene named M41 was retrieved mainly based on its high frequency in the muscle and low frequency in the liver. Structural analyses revealed that the AAVM41 capsid is a recombinant of AAV1, 6, 7, and 8 with a mosaic capsid surface and a conserved capsid interior. AAVM41 was then subjected to a side-by-side comparison to AAV9, the most robust AAV for systemic heart and muscle gene delivery; to AAV6, a parental AAV with strong muscle tropism. After i.v. delivery of reporter genes, AAVM41 was found more efficient than AAV6 in the heart and muscle, and was similar to AAV9 in the heart but weaker in the muscle. In fact, the myocardium showed the highest gene expression among all tissues tested in mice and hamsters after systemic AAVM41 delivery. However, gene transfer in non-muscle tissues, mainly the liver, was dramatically reduced. AAVM41 was further tested in a genetic cardiomyopathy hamster model and achieved efficient long-term delta-sarcoglycan gene expression and rescue of cardiac functions. Thus, direct in vivo panning of capsid libraries is a simple tool for the de-targeting and retargeting of viral vector tissue tropisms facilitated by acquisition of desirable sequences and properties.

Animals↗
PubMed1992

Gene transfer into mammalian somatic cells in vivo.

Direct gene transfer into mammalian somatic tissues in vivo is a developing technology with potential application for human gene therapy. During the past 2 years, extensive progress and numerous breakthroughs have been made in this area of research. Genetically engineered retroviral vectors have been used successfully to infect live animals, effecting foreign gene expression in liver, blood vessels, and mammary tissues. Recombinant adenovirus and herpes simplex virus vectors have been utilized effectively for in vivo gene transfer into lung and brain tissues, respectively. Direct injection or particle bombardment of DNA has been demonstrated to provide a physical means for in situ gene transfer, while carrier-mediated DNA delivery techniques have been extended to target specific organs for gene expression. These technological developments in conjunction with the initiation of the NIH human gene therapy trials have marked a milestone in developing new medical treatments for various genetic diseases and cancer. Various in vivo gene transfer techniques should also provide new tools for basic research in molecular and developmental genetics.

Animals↗
PubMed1986

[Phasmids. Properties and the use in genetic engineering. I. Construction of the phasmid vector].

A phasmid vector molecule designated pMYF11 has been constructed. The vector combines some useful features of plasmid and phage vector molecules. lambda pMYF11 is a hybrid of lambda 47.1 vector and pBR322 plasmid. CI- marker of pMYF11 is replaced with cI+ marker by recombination between the plasmid and prophage 434. The phasmid molecule can be used as a replacement vector for BamHI, HindIII, SalGI endonucleases. The maximum size of fragments to be cloned is 21 kilobase pairs. Positive selection for hybrid molecules is possible because of the Spi phenotype expression after replacement of the central HindIII or BamHI DNA fragment with foreign DNA. A library of Escherichia coli genes is constructed with the help of lambda pMYF11 as a vector molecule. A hybrid phage harboring genes of the proline operon is detected by means of complementation.

Bacteriophage lambda↗
PubMed1990

Analysis of the substrate binding sites of human galactosyltransferase by protein engineering.

An expression vector, pIN-GT, encoding the soluble form of beta 1,4-galactosyltransferase (GT) has been constructed from human GT cDNAs and the pIN-III-ompA2 expression vector. Escherichia coli strain SB221 harboring the pIN-GT plasmid produces and secretes a fusion protein consisting of the ompA signal and GT. The expression of GT was detected by assaying enzymatic activity as well as by Western blotting using anti-GT antibodies. The recombinant GT was purified to homogeneity by N-acetylglucosamine-Sepharose affinity chromatography. The NH2-terminal peptide sequence of purified GT confirmed the cleavage site of the fusion protein by bacterial signal peptidase. This expression system was utilized to produce mutant forms of GT in order to identify specific amino acids involved in substrate binding sites. Photoaffinity labeling of GT with UDP-galactose analog, 4-azido-2-nitrophenyluridylylpyrophosphate (ANUP), followed by cyanogen bromide (CNBr) cleavage revealed that ANUP bound to a fragment of GT composed of amino acid residues from Asp276 to Met328. Within this peptide segment, Tyr284, Tyr287, Tyr309, Trp310 and Trp312 were separately substituted into Gly and Tyr287 into Phe by site-directed mutagenesis. Enzymatic activity assay showed drastic reduction of the activity in all of the mutants except that Tyr287----Phe remained as active as wild-type GT. Kinetic studies of the mutated GT showed that Tyr284, Tyr309 and Trp310 are critically involved in the N-acetyglucosamine binding and Tyr309 is involved in UDP-galactose binding as well.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1987

Expression of human uterine tissue-type plasminogen activator in mouse cells using BPV vectors.

Human tissue-type plasminogen activator (t-PA) cDNA was cloned from uterine tissue and engineered in expression vectors for production in mouse C127 cells. The vectors consisted of the bovine papilloma virus-1 (BPV-1) genome and t-PA transcriptional unit with a mouse metallothionein (MT-1) promoter at the 5' end and MT-1 genomic sequences or SV40 early introns and polyadenylation signals at the 3' end. Analysis of the expression vectors transfected into cells revealed that t-PA is expressed 100- to 200-fold more with an intronless vector utilizing the SV40 polyadenylation signal than with other, intron-containing vectors. RNA analysis of stable cell lines indicated that t-PA expression levels correlated with mRNA abundance. DNA copy number and transcriptional rate of the MT-1 promoter remained constant in cell lines transformed by different BPV expression vectors. Uterine t-PA produced by recombinant DNA means was enzymatically active and similar in properties to Bowes melanoma t-PA.

Animals↗
PubMed1987

Use of vaccinia virus to express biopharmaceutical products.

Recent technological advancements have fostered the continued development of vaccinia virus as an efficient eukaryotic cloning and expression vector system. Genetically engineered vaccinia virus strains have been constructed for use (i) as recombinant vaccines for the prophylaxis of infectious disease, (ii) in producing significant quantities of biologically active polypeptide factors or enzymes, and (iii) as basic research tools with which to investigate primary structure-function relationships between proteins and their catalytic activities. This review examines the basic vaccinia vector system, its advantages and limitations, and current areas of research. As a specific example of the power and utility of this approach, attention is focused on the application of this technology to the field of neurobiology, specifically the use of recombinant vaccinia to study the expression, processing, and transport of cellular neuropeptides.

Cloning, Molecular↗
PubMedDate not supplied

[An computer information system for genetic engineering].

The article describes the vectors data base and the software for its use (the VECTOR-PC system). At present the prototype versions of data base and VECTOR-PC exist and are in test exploitation. The original data base entry format contains 17 main fields for specific genetic engineering information. The VECTOR-PC system includes programs for data base search and support, and also the "genetic engineering designer", which allows the user to design his own hypothetic structures from the objects of data base and to receive detailed information about them. The system is destined for IBM PC or compatible computers.

Genetic Engineering↗
PubMed1992

Expression and characterization of genetically engineered human immunodeficiency virus-like particles containing modified envelope glycoproteins: implications for development of a cross-protective AIDS vaccine.

Noninfectious human immunodeficiency virus type 1 (HIV-1) viruslike particles containing chimeric envelope glycoproteins were expressed in mammalian cells by using inducible promoters. We engineered four expression vectors in which a synthetic oligomer encoding gp120 residues 306 to 328 (amino acids YNKRKRIHIGP GRAFYTTKNIIG) from the V3 loop of the MN viral isolate was inserted at various positions within the endogenous HIV-1LAI env gene. Expression studies revealed that insertion of the heterologous V3(MN) loop segment at two different locations within the conserved region 2 (C2) of gp120, either 173 or 242 residues away from the N terminus of the mature subunit, resulted in the secretion of fully assembled HIV-like particles containing chimeric LAI/MN envelope glycoproteins. Both V3 loop epitopes were recognized by loop-specific neutralizing antibodies. However, insertion of the V3(MN) loop segment into other regions of gp120 led to the production of envelope-deficient viruslike particles. Immunization with HIV-like particles containing chimeric envelope proteins induced specific antibody responses against both the autologous and heterologous V3 loop epitopes, including cross-neutralizing antibodies against the HIV-1LAI and HIV-1MN isolates. This study, therefore, demonstrates the feasibility of genetically engineering optimized HIV-like particles capable of eliciting cross-neutralizing antibodies.

AIDS Vaccines↗
PubMed2024

Shedding light on Klebsiella pneumoniae virulence: Engineering of broad host range bioluminescence reporter vectors for transcriptional analysis in drug resistant pathogens.

In this work, we report the construction of four bacterial luciferase-based promoter probe vectors with an expanded set of selectable markers, designed to facilitate their use in antibiotic-resistant bacteria. These vectors contain the low-copy-number, broad-host-range pBBR origin of replication and an origin of transfer, allowing efficient conjugative transformation into various bacterial genera. The broad host range origin also enables their use in bacterial strains that harbor other plasmids, as the pBBR origin is compatible with a wide variety of other plasmid replication systems. The utility of these vectors was demonstrated by quantifying capsule gene expression in both classical and hypervirulent Klebsiella pneumoniae strains lacking tolC, which encodes the outer membrane pore protein for tripartite transport systems. Our results revealed that the tolC mutation reduced capsule gene expression, highlighting a critical role for tolC in K. pneumoniae pathobiology and the utility of bioluminescence for studying gene expression in real time. These new vectors provide a flexible platform for circumventing antibiotic resistance phenotypes and studying gene expression across diverse bacterial species, including strains containing additional plasmids.

Klebsiella pneumoniae↗
PubMed2026

Rab9 depletion enhances human adenovirus type 26 transduction efficiency through increased internalization and reduced late endosomal/lysosomal retention.

Understanding intracellular trafficking is central to decoding viral pathogenesis and engineering optimized viral vectors. How a virus or vector is routed through the endocytic pathway directly dictates its genome release, immune sensing, and overall transduction efficiency. Human adenovirus type 26 (HAdV-D26) presents a promising platform for vector design due to its low preexisting immunity, potent immune stimulation, scalable production, and versatile genetic engineering capacity. Although increasingly significant, the fundamental mechanisms governing HAdV-D26 intracellular trafficking are still not fully understood. Our study demonstrates that compared to well-described human adenovirus type 5 (HAdV-C5), HAdV-D26 undergoes prolonged intracellular trafficking, transiently localizing to early endosomes before residing in late endosomes/lysosomes for up to four hours post-infection. Inhibition of lysosomal acidification modestly enhances HAdV-D26 transduction efficiency, whereas blocking transport from early to late endosomes/lysosomes does not. Strikingly, Rab9 knockdown reduces HAdV-D26 late endosomal/lysosomal localization while increasing both virus internalization and genome delivery to the host cell nucleus. These findings indicate that late endosomal sorting pathways actively influence HAdV-D26 infection outcomes. By identifying a previously unappreciated role for Rab9 in adenovirus transduction, our results provide new mechanistic insight into HAdV-D26 intracellular trafficking, highlight serotype-specific differences in adenovirus entry pathways, and identify endosomal trafficking steps that may be targeted to improve adenoviral vector performance.

Humans↗