Philip A. Jennings

Delivery of nucleic acids

Nucleic acids have revolutionized biomedical research and have become indispensable research tools. In pharmaceutical development, nucleic acids are at present mostly used as diagnostic tools and for target validation (1– 3). Applications of microarrays and PCR, treatment with antisense oligonucleotides or small interfering RNA and breeding of knock-in/knock-out-models can be used for accurate diagnosis and biomarker detection, can improve insight into disease processes and can pinpoint pathways where treatments may interfere. Although the application of nucleic acids as therapeutics promises to be even more exciting, their role as clinically applied drugs is still modest. At present, two nucleic acid-based drugs (Vitravenei and Macugeni) are on the market (4). Both drugs are oligonucleotides. Macugeni is an extracellularly acting aptamer that functions as a growth factor decoy and Vitravenei is an intracellularly acting antisense molecule that inhibits a viral gene. Both oligonucleotides contain chemically modified backbones and are injected at the site of the pathology in the vitreous of the eye. This exemplifies the difficulties associated with the use of nucleic acids for therapeutic intervention, both regarding their physicochemical as well as their biological properties. The physicochemical properties of nucleic acids, with molecular weights ranging from 7 kDa for antisense oligonucleotides to over 1 MDa for plasmid DNA, and strong negative charge do not favor membrane passage. Only one class of nucleic acids, aptamers, can act extracellularly, which circumvents the need for cell membrane translocation. Conversely, all other classes need to interact with intracellular targets to be active. The problem is most prominent for plasmid DNA, which has the largest size of all proposed nucleic acid therapeutics and also needs to arrive inside the cell nucleus to be effective. Nuclear localization would in principle require passage through the nuclear pore for which the DNA-molecule is too large (5). These qualities at least partly explain why the marketed drugs are an aptamer and an antisense oligonucleotide. The biological properties also do not support their application as therapeutics. Nucleic acids are susceptible to the action of nucleases. Therefore the two marked oligonucleotides bear chemically modified backbones. In addition, nucleic acids are rapidly cleared from the body, either via glomerular filtration by the kidneys and excretion into the urine or by (scavenger) receptor uptake and intracellular degradation. Therefore, local injection at the site of the pathology is the preferred administration route for the clinically applied oligonucleotides. Despite these difficulties, nucleic acids still capture the mind of many pharmaceutical scientists as possible therapeutics. One of the most appealing properties is that a change in a disease target would in principle only require a change in the nucleic acid sequence to obtain a new drug. As the physicochemical properties like size and charge of the molecules remain the same, the same principles can be applied during the drug formulation steps for this new sequence. After successful formulation of the first nucleic acid drug it can be expected that subsequent formulations will follow more easily. In contrast, for small molecular weight drugs, lead compound identification requires high throughput screening, and drug formulation is dependent on the physicochemical and biological characteristics of the compound. Nevertheless, the difficult biopharmaceutical characteristics of nucleic acids put a lot of demands on the delivery systems that should compensate for these qualities by increasing stability against the action of nucleases, reducing excretion and uptake by non-target tissues and promoting target tissue interaction, target cell association, membrane translocation, and correct intracellular trafficking (6). The articles in this theme issue address this difficult drug formulation process. The group of Klibanov approached the problem of identifying suitable vectors for plasmid DNA delivery using a high-throughput-synthesis coupled to combinatorial chemistry approach. Their study is based on the cationic polymer poly(ethylene imine) (PEI). Experimental observations of their group and others indicate that PEI molecular weight is positively correlated with degree of transfection but also with severity of toxicity (7, 8). These observations provided the input for synthesizing small molecular weight PEI-derivatives that were cross-linked with oligo-acrylate esters. As many of the factors that contribute to degree of transfection and toxicity as well as the relative contribution of each factor to the overall transfection efficiency are unknown, the highthroughput synthesis approach likely provides a higher chance of finding successful polymers. Indeed, their results show that superior PEI-derivatives could be identified as compared to the presently used Fgolden standard_ 22 kD PEI both with respect to degree of transfection as well as toxicity both in vitro and in vivo. Most cationic polymers exhibit a molecular weight distribution. De Wolf et al. investigated the effects of fractionation of the biodegradable polymer poly(2-dimethylamino ethylamino)phosphazene (p(DMAEA)-ppz) into four different molecular weight fractions on in vitro/in vivo transfection of plasmid DNA and polymer-DNA-complex-

An engineered PGK promoter and lac operator-repressor system for the regulation of gene expression in mammalian cells

Previous reports have demonstrated that the Escherichia coli lac repressor can operate effectively in mammalian cells to repress expression of genes driven by modified viral or metallothionein (MT) promoters. We have developed a more general expression system using the promoter from the PGK1 gene (encoding murine 3-phosphoglycerate kinase) which is widely expressed in almost all cell types, including early embryonic and ES (embryonic stem) cells. Firstly, we engineered the lac repressor to include a nuclear localisation signal and placed it under control of the PGK1 promoter. Efficient nuclear localisation of the repressor was demonstrated by mobility-shift assays and immunofluorescence detection. For the target vectors, we modified the wild-type (wt) PGK1 promoter to include lac operator (lacO) sites for binding of the lac repressor and compared a number of different lacO positions and arrangements based on proximity to the native start points for transcription (tsp) and translation. In the absence of repressor, we observed reduced expression of the neo reporter gene for some placements of the lacO, but wt expression for placements near the tsp. When both target and repressor were present in the cells, we observed that the expression of neo could be strongly suppressed and reversibly regulated by induction with IPTG. In particular, for a promoter which contained two spaced lacO replacing native sequence around the major tsp, we observed 90-95% repression by the lac repressor for the neo reporter gene and up to 98% repression for the cat reporter gene. Efficient derepression by IPTG was observed in both cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning of a Lysobacter enzymogenes gene that encodes an arginyl endopeptidase (endoproteinase Arg-C)

Screening an expression library of Lysobacter enzymogenes DNA allowed us to clone a gene encoding a serine protease that cleaves synthetic substrates C-terminal to Arg and, to a lesser extent, Lys residues. The gene product, which shares sequence homology with the lysyl endopeptidases from L. enzymogenes and Achromobacter lyticus, consists of a signal sequence (24 residues), pro-region ( approximately 195 residues), and catalytic domain ( approximately 244 residues). Downstream of this gene is an open reading frame that lacks a promoter and appears to encode an inactive type I subtilase.