Antitumor and antimetastatic activity of ribozymes targeting the messenger RNA of vascular endothelial growth factor receptors.

Chemically stabilized hammerhead ribozymes are nuclease-resistant, RNA-based oligonucleotides that selectively bind and cleave specific target RNAs. Due to their potential for specifically inhibiting gene expression, ribozymes are being investigated for therapeutic applications as well as for the elucidation of gene function. In particular, we have investigated ribozymes that target the mRNA of the vascular endothelial growth factor (VEGF) receptors because VEGF signaling is an important mediator of tumor angiogenesis and metastasis. Here we report pharmacodynamic studies testing anti-Flt-1 (VEGFR-1) and anti-KDR (VEGFR-2) ribozymes in animal models of solid tumor growth and metastasis. Ribozymes targeting either Flt-1 or KDR significantly inhibited primary tumor growth in a highly metastatic variant of Lewis lung carcinoma. However, only treatment with the anti-Flt-1 ribozyme resulted in a statistically significant and dose-dependent inhibition of lung metastasis in this model. The anti-Flt-1 ribozyme was then tested in a xenograft model of human metastatic colorectal cancer in which significant inhibition of liver metastasis was observed. Taken together, these data represent the first demonstration that synthetic ribozymes targeting VEGF receptor mRNA reduced the growth and metastasis of solid tumors in vivo.

Bioactivity of anti-angiogenic ribozymes targeting Flt-1 and KDR mRNA.

Vascular endothelial growth factor (VEGF) and its receptors Flt-1 and KDR play important roles in physiological and pathological angiogenesis. Ribozymes that target the VEGF receptor mRNAs were developed and their biological activities in cell culture and an animal model were assessed. Ribozymes targeting Flt-1 or KDR mRNA sites reduced VEGF-induced proliferation of cultured human vascular endothelial cells and specifically lowered the level of Flt-1 or KDR mRNA present in the cells. Anti- Flt-1 and KDR ribozymes also exhibited anti-angiogenic activity in a rat corneal pocket assay of VEGF-induced angiogenesis. This report illustrates the anti-angiogenic potential of these ribozymes as well as their value in studying VEGF receptor function in normal and pathophysiologic states.

Interactions of oligonucleotide analogs containing methylphosphonate internucleotide linkages and 2'-O-methylribonucleosides.

The interactions of oligonucleotide analogs, 12-mers, which contain deoxyribo- or 2'-O-methylribose sugars and methylphosphonate internucleotide linkages with complementary 12-mer DNA and RNA targets and the effect of chirality of the methylphosphonate linkage on oligomer-target interactions was studied. Oligomers containing a single Rp or Sp methylphosphonate linkage (type 1) or oligomers containing a single phosphodiester linkage at the 5'-end followed by 10 contiguous methylphosphonate linkages of random chirality (type 2) were prepared. The deoxyribo- and 2'-O-methylribo- type 1 12-mers formed stable duplexes with both the RNA and DNA as determined by UV melting experiments. The melting temperatures, Tms, of the 2'-O-methylribo-12-mer/RNA duplexes (49-53 degrees C) were higher than those of the deoxyribo-12mer/RNA duplexes (31-36 degrees C). The Tms of the duplexes formed by the Rp isomers of these oligomers were approximately 3-5 degrees C higher than those formed by the corresponding Sp isomers. The deoxyribo type 2 12-mer formed a stable duplex, Tm 34 degrees C, with the DNA target and a much less stable duplex with the RNA target, Tm < 5 degrees C. In contrast, the 2'-O-methylribo type 2 12-mer formed a stable duplex with the RNA target, Tm 20 degrees C, and a duplex of lower stability with the DNA target, Tm < 5 degrees C. These results show that the previously observed greater stability of oligo-2'-O-methylribonucleotide/RNA duplexes versus oligodeoxyribonucleotide/RNA duplexes extends to oligomers containing methylphosphonate linkages and that the configuration of the methylphosphonate linkage strongly influences the stability of the duplexes.

Synergistic antiviral activities of oligonucleoside methylphosphonates complementary to herpes simplex virus type 1 immediate-early mRNAs 4, 5, and 1.

An oligonucleoside methylphosphonate (ONMP) complementary to the splice acceptor site of immediate-early (IE) pre-mRNAs 4 and 5 (IE4,5SA) inhibits herpes simplex virus type 1 (HSV-1) growth in vitro and in infected animals. The antiviral effect appears to be due to inhibition of IE pre-mRNA 4 and 5 splicing and/or IE4 gene expression (M. Kulka, M. Wachsman, S. Miura, R. Fishelevich, P. S. Miller, P. O. P. Ts'o, and L. Aurelian, Antiviral Res. 20:115-130, 1993). We describe the potentiation of antiviral activity when we targeted two IE genes with different ONMPs. A psoralen derivative of an ONMP complementary to the IE mRNA 1 (IE1) translation initiation site (IE1TI) covalently bound a 2.8-kb transcript that hybridized with a 20-base oligonucleotide complementary to the 5' leader sequence of IE1 but not a 20-base oligonucleotide complementary to the first intron of IE1. IE1TI inhibited IE1 gene expression and virus replication in cells infected with HSV-1 in vitro. Inhibition was specific because it was not observed with oligomers mutated in two (IE1TImu1) or four (IE1TImu2) central residues or in cells infected with an IE1 deletion mutant (HSV-1 dl1403). IE1TI potentiated the antiviral activity of IE4,5SA (synergistic effect), while potentiation was not observed when IE4,5SA was mixed with IE1TImu1. A similar synergistic effect was seen when IE1TI was mixed with an ONMP complementary to the translation initiation site of IE mRNA 4 but not with an ONMP complementary to the translation initiation site of IE mRNA 5. These findings suggest that synergistic antiviral activity is mediated by targeting at least two IE genes (IE1 and IE4).

A structured literature review of treatment for localized prostate cancer. Prostate Disease Patient Outcome Research Team.

OBJECTIVE: We performed a structured literature review to define the clinical course of localized prostate cancer, the effectiveness of radical surgery and radiation therapy, and treatment complications. ARTICLE SELECTION: We identified more than 1600 English-language, MEDLINE referenced articles for 1966 through 1991. All but 144 were excluded because they lacked primary data, involved fewer than 15 patients, or described neither the course of the disease nor treatment complications. DATA SYNTHESIS: In these 144 articles, persistent genitourinary complications were more common after radical surgery than after external-beam radiation. Radiation resulted in a higher incidence of bowel problems. The median annual risks for the development of distant metastases and cancer-related death were 2.6% and 1.0%, respectively. Because tumor grade was correlated with metastases (Spearman correlation r = .56) and cancer mortality (r = .31), controlling for grade was necessary before we could compare the effectiveness of treatments for these outcomes. However, stratification by grade of malignancy was available in only nine of the patient series describing metastatic rates and in seven describing cancer-related mortality. Furthermore, in the patient series that described prostate cancer-related metastatic rates, 48% neglected to identify patients unavailable for follow-up, 92% did not stratify patients by age, and only 48% stratified patients by the extent of disease at treatment. CONCLUSIONS: Although we were able to compare complications of treatments, we were unable to determine treatment effectiveness for localized prostate cancer because of methodologic inadequacies in the literature we reviewed. Until better scientific evidence is available, patients and their physicians cannot make informed choices based on knowledge of the benefits of radical prostatectomy, radiation, or watchful waiting.

Triplex formation by oligodeoxyribonucleotides involving the formation of X.U.A triads.

The stabilities of oligodeoxyribonucleotide triplexes containing a single pyrimidine-purine base pair, which interrupts an otherwise purine-pyrimidine base pair motif, were studied by UV melting experiments. The oligomer systems consisted of an oligodeoxyribonucleotide target duplex d-GAAGAAAAAAYAAAA/d-TTTTZTTTTTTCTTC, I.II(Y.Z), or d-GAAGAAAAAGUGAAA/d-TTTCACTTTTTCTTC, IV.V(U.A), where Y.Z is C.G, T.A, or U.A and U is deoxyuridine. The third strand oligodeoxyribopyrimidine was d-CTTCTTTTTTXTTTT, III(X), or d-CTTCTTTTTCXCTTT, VI(X), where C is 5-methyldeoxycytidine. Triplexes were observed in the system III.I.II(X.C.G) when X was T or U. This may involve formation of T. or U.C.G triads in which the 4-carbonyl of T or U serves as a hydrogen bond acceptor for the N4-amino group of C. Triplex formation between III(X) and I.II(T.A) was only observed when X was G. In contrast to T.A or C.G, it appears a U.A base pair in the duplex target is a much more versatile participant in triad formation. Thus, stable triplexes were observed in III.I.II(X.U.A) and in VI.IV.V(X.U.A) when X was C, C, T, or U. The formation of a T.U.A or U.U.A triad can occur if the T or U of III translates approximately 1.4 A into the major groove, thereby allowing the 3-NH of T or U to donate a hydrogen bond to the 4-carbonyl oxygen of U in the duplex. Formation of C. or C.U.A base triads could involve formation of a single hydrogen bond between the third strand N4-amino group of C or C and the 4-carbonyl group of U of the target.(ABSTRACT TRUNCATED AT 250 WORDS)

Recognition of a guanine-cytosine base pair by 8-oxoadenine.

Two oligodeoxyribonucleotides, d-CTTCTTTTTTATTTT, I(A), and d-ATTATTTTTTATTTT, II(A), where C is 5-methylcytosine and A is 8-oxoadenine, were prepared and their interactions with the duplex d-GAAGAAAAAAYAAAA/d-TTTTZTTTTTTCTTC, III.IV(Y.Z), were studied. Oligomers I(A) and II(A) each form triplexes with III.IV(G.C) at temperatures below 20 degrees C as shown by continuous variation experiments, melting experiments, and circular dichroism (CD) spectroscopy. The CD spectra of these triplexes are almost identical to those formed by I(C) and II(C), oligomers which contain cytosine in place of 8-oxoadenine. This suggests that the 8-oxoadenine-containing triplexes have conformations which are very similar to those of the cytosine-containing triplexes. The melting temperature (Tm) for dissociation of the third strand of triplex II.III.IV(A.G.C) is 22 degrees C at pH 7.0 and 8.0, whereas the Tm of the corresponding transition in triplex II.III.IV(C.G.C) decreases from 28 degrees C at pH 7.0 to 17 degrees C at pH 8.0. The pH dependence of the Tm in the latter triplex reflects the necessity of protonating the N-3 of cytosine in order for it to form two hydrogen bonds with G of the G.C base pair. It appears that the keto form of 8-oxoadenine can potentially form two hydrogen bonds with the N-7 and O-6 atoms of G of the G.C base pair, when the 8-oxoadenine is in the syn conformation and in contrast to cytosine does not require protonation of the base. Oligomer I(A) does not form triplexes with III.IV(Y.Z) when Y.Z is A.T or T.A.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective modification of cytosines in oligodeoxyribonucleotides.

A single deoxycytidine residing in an oligodeoxyribonucleotide which also contains 5-methyldeoxycytidines can be selectively derivatized with various alkylamines by sodium bisulfite-catalyzed transamination. Selective transamination results because 5-methylcytosine, unlike cytosine, does not form a bisulfite adduct. When the reaction is carried out at pH 7.1, transamination in the oligomer appears to occur to greater than 95% with little or no deamination. This procedure has been used to introduce aminoalkyl or carboxyalkyl side chains at the N4-position of a deoxycytidine in oligonucleotides. These side chains contain potentially reactive amine or carboxy groups which could serve as a sites for further conjugation of the oligomer with a variety functional groups. Oligonucleotides which carry these side chain form duplexes and triplexes with appropriate complementary single-stranded or double-stranded oligodeoxyribonucleotide target molecules. The stabilities of the duplexes are similar to those formed by unmodified oligomers, whereas the stability of the triplexes is approximately 18 degrees C lower than that formed by unmodified oligomers.

Antisense inhibition of ras p21 expression that is sensitive to a point mutation.

Many genetic disorders result from a single point mutation, and many tumor oncogenes have been found to be altered by a point mutation. The ability to inhibit selectively the expression of the mutated form of a protein without affecting its normal counterpart is central to many therapeutic strategies, since the normal protein may serve indispensable functions. Antisense oligonucleoside methylphosphonates and their psoralen derivatives directed at either normal human Ha-ras p21 or ras p21 that is mutated at a single base in codon 61 have been examined for their efficacy and specificity as inhibitors of p21 expression. Mixed cultures of cells expressing both forms of p21 were treated with the antisense oligomer complementary to the normal p21 or with the antisense oligomer complementary to the point-mutated p21. Each of the antisense oligomers specifically inhibited expression of only the form of ras p21 to which it was completely complementary and left the other form of p21 virtually unaffected.

Photochemical cross-linking of psoralen-derivatized oligonucleoside methylphosphonates to rabbit globin messenger RNA.

Antisense oligodeoxyribonucleoside methylphosphonates targeted against various regions of mRNA or precursor mRNA are selective inhibitors of mRNA expression both in cell-free systems and in cells in culture. The efficiency with which methylphosphonate oligomers interact with mRNA, and thus inhibit translation, can be considerably increased by introducing photoactivatable psoralen derivatives capable of cross-linking with the mRNA. Oligonucleoside methylphosphonates complementary to coding regions of rabbit alpha- or beta-globin mRNA were derivatized with 4'-(aminoalkyl)-4,5',8-trimethylpsoralens by attaching the psoralen group to the 5' end of the oligomer via a nuclease-resistant phosphoramidate linkage. The distance between the psoralen group and the 5' end of the oligomer can be adjusted by changing the number of methylene groups in the aminoalkyl linker arm. The psoralen-derivatized oligomers specifically cross-link to their complementary sequences on the targeted mRNA. For example, an oligomer complementary to nucleotides 56-67 of alpha-globin mRNA specifically cross-linked to alpha-globin mRNA upon irradiation of a solution of the oligomer and rabbit globin mRNA at 4 degrees C. Oligomers derivatized with 4'-[[N-(2-amino-ethyl)amino]methyl]-4,5',8-trimethylpsoralen gave the highest extent of cross-linking to mRNA. The extent of cross-linking was also determined by the chain length of the oligomer and the structure of the oligomer binding site. Oligomers complementary to regions of mRNA that are sensitive to hydrolysis by single-strand-specific nucleases cross-linked to an approximately 10-30-fold greater extent than oligomers complementary to regions that are insensitive to nuclease hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)