OvaRex (AltaRex).

AltaRex is developing OvaRex (B43.13), a monoclonal antibody vaccine, for the potential treatment of ovarian cancer. Additionally, immunotherapy may be possible in other cancers expressing the CA125 antigen, such as breast and lung cancers. AltaRex plans to file a BLA for OvaRex with the FDA in late 2001 and for Canadian and European regulatory submissions thereafter, with possible commercialization in 2002 [365081,377828,398528]. In December 2000, AltaRex engaged US Oncology to participate in the company's phase II trial for the 'watchful waiting' stage of ovarian cancer. The US Oncology relationship will bring over 20 satellite sites to the OvaRex study, in addition to 13 sites that had already begun enrolling patients [394604,384676,365081]. In 1997, AltaRex commenced a multicenter, placebo-controlled, double-blind, randomized phase IIb trial in the US for advanced ovarian cancer [230067,331744,344248]. In the following year, AltaRex submitted the IND to the FDA for this trial [303667]. Complete analysis of the data is expected by the first half of 2001 [291312,310071,344248]. If an NDA is filed before the end of 2001, approval by mid-2002 is possible [344248]. OvaRex has been awarded Fast Track designation for advanced ovarian cancer [310071] and Orphan Drug status in the US [230067].

INGN-201. Introgen Therapeutics.

In April 2001, Aventis and Introgen signed a letter of intent to restructure their collaboration arrangement, giving Introgen responsibility for the worldwide development of all p53 programs under the existing collaboration and obtain exclusive worldwide commercial rights to p53-based gene therapy products, including INGN-201. In February 2001, Introgen was awarded US-06194191 for the commercial production of adenovirus vectors. In November 2000, the company and the University of Texas System were issued with US-06143290, entitled 'Recombinant p53 adenovirus methods and compositions', further solidifying the company's current p53 patent portfolio. In June 2000, the University of Texas System was awarded US-06069134, entitled, 'Methods and compositions comprising DNA damaging agents and p53.' This is the second US patent to be issued that is equivalent to WO-09528948. In May 1998, US-05747469, entitled 'Methods and compositions comprising DNA damaging agents and p53', was awarded to the Board of Regents of the University of Texas. This patent covers the use of the p53 gene in combination with chemotherapeutic agents, radiation therapies or other agents, which damage the DNA of cancer cells. It is one of several intellectual properties licensed to Introgen through an agreement with the MD Anderson Cancer Center.

Cellular proteins prevent antisense phosphorothioate oligonucleotide (SdT18) to target sense RNA (rA18): development of a new in vitro assay.

There are numerous indications that the "antisense" mechanism alone cannot account for the observed effects in living cells. Despite that, interactions between antisense oligonucleotides (ASO) and cellular proteins are usually not considered. In this work, we have tested the ability of antisense phosphorothioate (SdT) oligonucleotides and natural deoxyoligonucleotides (dT) for their ability to interact with target RNA in the presence of cellular proteins. We show that the affinity for cellular proteins is an essential factor that determines the success of RNA targeting. We have used a simple nuclease digestion assay to detect RNA/ASO hybrid formation in the presence of proteins. The results show the inability of a phosphorothioate oligonucleotide (SdT18) to reach the target RNA (rA18) in vitro in the presence of proteins. However, if proteins are absent, the RNA targeting was successful, as is usual in in vitro assays. Note that the target RNA concentration exceeded physiological values by several orders of magnitude while the crude protein extract was 20-fold diluted in the reaction tube. This finding is compatible with the notion that therapeutic properties of phosphorothioates could largely derive from a so-called "aptamer" effect.

Differential behavior of curved DNA upon untwisting.

We have synthesized DNA segments with different handedness, twisting and radii of curvature, and have analyzed the effect of untwisting on them. The results indicate that the dynamic behavior of curved DNA upon untwisting is strongly determined by the initial sequence-dependent DNA trajectory. In particular, DNA with the same radii but with opposite handedness of superhelix twisting can show very different conformational responses to ethidium bromide untwisting. Upon treatment with ethidium bromide, right-handed superhelixes decrease their twist and increase the planarity of the superhelix, while left-handed superhelixes increase twisting and decrease their degree of planarity.

Trinucleotide models for DNA bending propensity: comparison of models based on DNaseI digestion and nucleosome packaging data.

DNaseI digestion studies (Brukner et al, EMBO J 14, 1812-1818 1995) and nucleosomebinding data (Satchwell et al, J. Mol. Biol. 191, 639-659 1986, Goodsell and Dickerson, Nucleic trinucleotides. A detailed comparison of the two models suggests that while both of them represent improvements with respect to dinucleotide based descriptions, the individual trinucleotide parameters are not highly correlated (linear correlation coefficient is 0.53), and a number of motifs such as TA-elements and CCA/TGG motifs are more realistically described in the DNaseI-based model. This may be due to the fact that the DNaseI-based model does not rely on a static geometry but rather captures a dynamic ability of ds DNA to bend towards the major grove. Future refinement of both models of both models on larger experimental data sets is expected to further improve the prediction of macroscopic DNA-curvature.

Sequence-dependent bending propensity of DNA as revealed by DNase I: parameters for trinucleotides.

Structural parameters characterizing the bending propensity of trinucleotides were deduced from DNase I digestion data using simple probabilistic models. In contrast to dinucleotide-based models of DNA bending and/or bendability, the trinucleotide parameters are in good agreement with X-ray crystallographic data on bent DNA. This improvement may be due to the fact that the trinucleotide model incorporates more sequence context information than do dinucleotide-based descriptions.

Mutational analysis of DNase I-DNA interactions: design, expression and characterization of a DNase I loop insertion mutant with altered sequence selectivity.

A mutant of bovine pancreatic DNase I containing two additional residues in a loop next to C173 has been expressed in Escherichia coli, purified and characterized biochemically. Modelling studies suggest that the inserted arginine and glutamate side chains of the modified loop sequence C173-R-E-G-T-V176 could contact the bases 3' to the cleaved bond in the major groove of a bound DNA, and that up to 10 bp could interact with the enzyme and potentially influence its cutting rate. The loop insertion mutant has an 800-fold lower specific activity than wild-type and shows overall cleavage characteristics similar to bovine pancreatic DNase I. Compared with the wild-type enzyme, the mutant shows a strongly enhanced preference for cutting the inverted repeat: (formula: see text) or close variants thereof. Unexpectedly for a minor groove binding protein, the preferred cutting sites in opposite strands are staggered by 1 bp in the 5' direction, causing the cleavage of a TA and a TT step, respectively. This finding demonstrates that the sequence context is relatively more important for the cutting frequency than the nature of the dinucleotide step of the cleaved bond, and clearly shows that base recognition is involved in determining the sequence selectivity of the mutant. The importance of the sequence 5' to the cleaved bond for the cutting rate suggests that the additional major groove contacts may require a distortion of the DNA associated with a higher energy barrier, resulting in an increased selectivity for flexible DNA sequences and a lower overall activity of the mutant enzyme.

Physiological concentration of magnesium ions induces a strong macroscopic curvature in GGGCCC-containing DNA.

The bending propensity of non-A/T DNA sequence elements is well known, but helical phasing/gel mobility experiments fail to reveal an intensive macroscopic curvature if A/T tracts are not present in the sequence. Recent X-ray data prove on the other hand that a GGCC element is intrinsically curved toward the major groove, which seemingly contradicts the fact that macroscopic curvature at GGGCCC elements is hardly detectable with a conventional gel mobility assay. Here we show that GGGCCC containing DNA, with no A/T tracts in the sequence context, has a detectable, strong gel mobility anomaly only in the presence of divalent ions (10 mM Mg2+ or Ca2+, 1 mM Zn2+). Metal ions increase the gel mobility anomaly in A/T tracts as well, but the effect is substantially stronger for GGGCCC than for the rigid A/T tracts. Our data suggest that metal ions change the sequence-dependent dynamic features of DNA; on the other hand, there is no evidence of twist-mediated change of the planarity of curvature in the presence of metal ions. The results show that near-physiological concentrations of divalent cations (10 mM MgCl2) have a strong and differential effect on various sequence elements, so that the current picture of sequence-dependent DNA curvature is changed not only in a quantitative, but also in a qualitative sense.

Evidence for opposite groove-directed curvature of GGGCCC and AAAAA sequence elements.

The repetitive sequence (AGGGCCCTAGAGGGGCCC-TAG)n was previously shown to be curved by gel mobility assays. Here we show, using hydroxy radical/DNase I digestion and differential helical phasing experiments that the curvature is directed towards the major groove and is located in the GGGCCC, but not the CTAGAG segments. The effect of the GC step in the context of the GGGCCC motif is apparently about as large as that of AA/TT, i.e. enough to cancel the macroscopic curvature of helically phased A-tracts. These data are in agreement with positive roll-like curvature of the GCC/GGC motif, predicted from nucleosome packing data and the 3D structure of the GGGGCCCC octamer, but they are not in agreement with the dinucleotide-based roll angle values predicted for AG/CT, TA, GG/CC and GC steps. Our results thus indicate the importance of interactions beyond the dinucleotide steps in predictive models of DNA curvature.

Curved DNA without AA/TT dinucleotide step.

The evidence is accumulating that dinucleotide steps other than AA/TT affect DNA flexure of AnTm (m + n greater than = 4) containing fragments. However, it is not clear whether macroscopic DNA flexure without AA/TT steps might occur. In this paper we demonstrate the anomaly in electrophoretic mobility of non AA/TT repetitive DNA sequences which is a function of sequence phasing. Therefore, our results show that PyPu (TA) and AG/CT steps, angulary separated by close to 180 degrees from Pu/Py (GC) and GG/CC steps, bend DNA, even in the absence of AnTm tracts.

Sequence-dependent structural variations of DNA revealed by DNase I.

Two global helix parameters important for DNA-DNase I interaction are the geometry of the minor groove and the DNA stiffness that resists bending toward major groove. Thus, local averaging of P-O3' bonds cutting frequencies (InP) reflects global helix parameters revealed by DNase I. Using the approximation that locally averaged InP values depend only on the type of the dinucleotide steps involved in the region of interaction, we calculated the collective contribution (sigma Dd) for ten different dinucleotide steps. Our results suggest that, at the first approximation, global varying helix parameters revealed by DNase I, might be predicted from sequence. Obtained sigma Dd function can be used as a sequence-dependent measure of protein-induced DNA flexure in the direction towards the major groove, which is usually connected to widening of the minor groove. In the course of analysis of Mg2+ and Mn2+ dependent DNase I digestions, no significant difference was found, in spite of the supposed differences in enzyme activity. These results suggest that if the second Mn2(+)-dependent active site exists, its activity is lower than that of the first one.

Sequencing of megabase plus DNA by hybridization: theory of the method.

A mismatch-free hybridization of oligonucleotides containing from 11 to 20 monomers to unknown DNA represents, in essence, a sequencing of a complementary target. Realizing this, we have used probability calculations and, in part, computer simulations to estimate the types and numbers of oligonucleotides that would have to be synthesized in order to sequence a megabase plus segment of DNA. We estimate that 95,000 specific mixes of 11-mers, mainly of the 5'(A,T,C,G)(A,T,C,G)N8(A,T,C,G)3' type, hybridized consecutively to dot blots of cloned genomic DNA fragments would provide primary data for the sequence assembly. An optimal mixture of representative libraries in M13 vector, having inserts of (i) 7 kb, (ii) 0.5 kb genomic fragments randomly ligated in up to 10-kb inserts, and (iii) tandem "jumping" fragments 100 kb apart in the genome, will be needed. To sequence each million base pairs of DNA, one would need hybridization data from about 2100 separate hybridization sample dots. Inevitable gaps and uncertainties in alignment of sequenced fragments arising from nonrandom or repetitive sequence organization of complex genomes and difficulties in cloning "poisonous" sequences in Escherichia coli, inherent to large sequencing by any method, have been considered and minimized by choice of libraries and number of subclones used for hybridization. Because it is based on simpler biochemical procedures, our method is inherently easier to automate than existing sequencing methods. The sequence can be derived from simple primary data only by extensive computing. Phased experimental tests and computer simulations increasing in complexity are needed before accurate estimates can be made in terms of cost and speed of sequencing by the new approach. Nevertheless, sequencing by hybridization should show advantages over existing methods because of the inherent redundancy and parallelism in its data gathering.