The 50th anniversary of gene therapy: beginnings and present realities.

Applying my experience in microbial genetics, especially in the genetic transformation/transduction of Bacilus subtilis bacteria, I decided around 1956 to develop a similar system for eukaryotic, especially human cell cultures. I believed it would permit the development of clinical applications for replacing defective genes to treat or cure some of the genetic diseases.

Isolation of single, intact chromosomes from single, selected ovarian cancer cells for in situ hybridization and sequencing.

The first step towards effective therapy of cancer is to reveal molecular profiles of all cell clones propelling tumor growth. The specific aim of this project was to develop a technology helping us to isolate patient's single, living cells based upon their cancer-specific, cell surface biomarkers, to reveal their molecular profiles, and to isolate, from these selected cells, intact chromosomes for in situ hybridization (FISH) and for next generation sequencing (NGS). We attained this aim, while probing the cells from the ovarian cancer patients. Ovarian cancer is the most deadly of all gynecological cancers. In most of the patients with the advanced stages of this cancer, the gene for epidermal growth factors receptor (EGFR) is mutated, as the deletion variant III, resulting in the truncated transcripts and products. From these patients, we collected cells from peritoneal fluid, blood, lymph, and biopsies. We genetically engineered fluorescent and superparamagnetic single chain variable fragments (scFvs) targeting EGFRwt and EGFRvIII. Using these scFvs, we isolated single, living ovarian cancer cells and analyzed their transcripts and products. We further genetically engineered scFv targeting dsDNA. Using these scFvs, we isolated the entire single, intact chromosomes from the selected, single ovarian cancer cells for NGS and for liquid phase FISH. This novel work-flow opens new routes not only for molecular profiling of the entire spectrum of cancer cell clones in the diagnosed patient, one cell clone at a time, but also for manufacturing targeted contrast for in vivo imaging and for designing and guiding targeted delivery of therapeutic genes in cancer therapy.

Gram negative shuttle BAC vector for heterologous expression of metagenomic libraries.

Bacterial artificial chromosome (BAC) vectors enable stable cloning of large DNA fragments from single genomes or microbial assemblages. A novel shuttle BAC vector was constructed that permits replication of BAC clones in diverse Gram-negative species. The "Gram-negative shuttle BAC" vector (pGNS-BAC) uses the F replicon for stable single-copy replication in E. coli and the broad-host-range RK2 mini-replicon for high-copy replication in diverse Gram-negative bacteria. As with other BAC vectors containing the oriV origin, this vector is capable of an arabinose-inducible increase in plasmid copy number. Resistance to both gentamicin and chloramphenicol is encoded on pGNS-BAC, permitting selection for the plasmid in diverse bacterial species. The oriT from an IncP plasmid was cloned into pGNS-BAC to enable conjugal transfer, thereby allowing both electroporation and conjugation of pGNS-BAC DNA into bacterial hosts. A soil metagenomic library was constructed in pGNS-BAC-1 (the first version of the vector, lacking gentamicin resistance and oriT), and recombinant clones were demonstrated to replicate in diverse Gram-negative hosts, including Escherichia coli, Pseudomonas spp., Salmonella enterica, Serratia marcescens, Vibrio vulnificus and Enterobacter nimipressuralis. This shuttle BAC vector can be utilized to clone genomic DNA from diverse sources, and then transfer it into diverse Gram-negative bacterial species to facilitate heterologous expression of recombinant pathways.

Novel sequencing strategy for repetitive DNA in a Drosophila BAC clone reveals that the centromeric region of the Y chromosome evolved from a telomere.

The centromeric and telomeric heterochromatin of eukaryotic chromosomes is mainly composed of middle-repetitive elements, such as transposable elements and tandemly repeated DNA sequences. Because of this repetitive nature, Whole Genome Shotgun Projects have failed in sequencing these regions. We describe a novel kind of transposon-based approach for sequencing highly repetitive DNA sequences in BAC clones. The key to this strategy relies on physical mapping the precise position of the transposon insertion, which enables the correct assembly of the repeated DNA. We have applied this strategy to a clone from the centromeric region of the Y chromosome of Drosophila melanogaster. The analysis of the complete sequence of this clone has allowed us to prove that this centromeric region evolved from a telomere, possibly after a pericentric inversion of an ancestral telocentric chromosome. Our results confirm that the use of transposon-mediated sequencing, including positional mapping information, improves current finishing strategies. The strategy we describe could be a universal approach to resolving the heterochromatic regions of eukaryotic genomes.

Copy-control pBAC/oriV vectors for genomic cloning.

The use of the improved BAC system for cloning genomic DNA and library constructions is described. This system retains all the advantages of the original BACs but, in addition, permits, on command, amplification of the BAC plasmids and cloned DNA. This system consists of (1) plasmid pBAC/oriV containing an additional replication origin, oriV, and (2) a host carrying the up-mutants of the trfA replicator gene expressed from the l-arabinose-inducible Para promoter. The pBAC/oriV clones are always maintained in the single-copy state, but if more DNA is required, they could be amplified up to 100-fold, depending on the size of the cloned insert.

Copy-control tightly regulated expression vectors based on pBAC/oriV.

A novel type of expression vectors with a dual regulation of both the plasmid copy number and gene expression, is described. The most important and beneficial feature of these vectors is that when they are not induced, they are maintained as a single-copy plasmid, and therefore, any residual expression is much more tightly regulated than for the conventional multicopy expression vectors. The simplest version of these copy-control expression vectors is based on the pBAC/oriV plasmid that carries the trfA up-mutant gene under control of the l-arabinose-inducible Para promoter (araC-PBAD). The same promoter controls expression of a gene cloned into MCS. Thus, addition of the inducer (l-arabinose) simultaneously turns on amplification of the plasmid and expression of the cloned gene. Net result is about a 50,000-fold increase in the cloned gene expression. However, when not induced, background expression level is very low, which is important for the maintenance of any "toxic" genes. This vector could be used in most E. coli hosts. Similar versions of the described vector employ the rhamnose-inducible Prha promoter (rhaS-Prha). Other expression systems allow independent regulation of the plasmid amplification and of the cloned gene expression, and some also use the PLtetO-1 promoter. Copy-control expression vector pETcoco, based on the pT7lacO promoter, is commercially available.

A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes.

A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.

Conditionally amplifiable BACs: switching from single-copy to high-copy vectors and genomic clones.

The widely used, very-low-copy BAC (bacterial artificial chromosome) vectors are the mainstay of present genomic research. The principal advantage of BACs is the high stability of inserted clones, but an important disadvantage is the low yield of DNA, both for vectors alone and when carrying genomic inserts. We describe here a novel class of single-copy/high-copy (SC/HC) pBAC/oriV vectors that retain all the advantages of low-copy BAC vectors, but are endowed with a conditional and tightly controlled oriV/TrfA amplification system that allows: (1) a yield of ~100 copies of the vector per host cell when conditionally induced with L-arabinose, and (2) analogous DNA amplification (only upon induction and with copy number depending on the insert size) of pBAC/oriV clones carrying >100-kb inserts. Amplifiable clones and libraries facilitate high-throughput DNA sequencing and other applications requiring HC plasmid DNA. To turn on DNA amplification, which is driven by the oriV origin of replication, we used copy-up mutations in the gene trfA whose expression was very tightly controlled by the araC-P(araBAD) promoter/regulator system. This system is inducible by L-arabinose, and could be further regulated by glucose and fucose. Amplification of DNA upon induction with L-arabinose and its modulation by glucose are robust and reliable. Furthermore, we discovered that addition of 0.2% D-glucose to the growth medium helped toward the objective of obtaining a real SC state for all BAC systems, thus enhancing the stability of their maintenance, which became equivalent to cloning into the host chromosome