The role of area deprivation index in health care disruptions among cancer survivors during the SARS-CoV-2 pandemic.

OBJECTIVE: To examine the associations between demographic/medical and geographic factors with follow-up medical care and health-related quality of life (HRQoL) among cancer survivors during the SARS-CoV-2 pandemic. STUDY DESIGN: Cross-sectional survey. METHODS: An online survey was sent to cancer survivors between May 2020 and January 2021, exploring their experience with SARS-CoV-2, follow-up care, and HRQoL. PolicyMap was used to geocode home addresses. Both geographic and demographic/medical factors were examined for their associations with SARS-CoV-2 experience, follow-up care, and HRQoL (FACT-G7). RESULTS: Geographic data were available for 9651 participants. Patients living in the highest area deprivation index (ADI) neighborhoods (most deprived) had higher odds of avoiding in-person general (odds ratio [OR] = 7.20; 95% confidence interval [CI] = 2.79-18.60), cancer (OR = 8.47; 95% CI = 3.73-19.30), and emergency (OR = 14.2; 95% CI = 5.57-36.30) medical care, as well as lower odds of using telemedicine (OR = 0.61; 95% CI = 0.52-0.73) compared to the lowest ADI group. Race/ethnicity was not associated with follow-up care after controlling for ADI. The effect of ADI on HRQoL was generally in the expected direction, with higher ADI being associated with worse HRQoL. CONCLUSIONS: ADI influenced follow-up medical care more than age, race/ethnicity, or health insurance type. Healthcare providers and institutions should focus on decreasing barriers to in-person and telemedicine health care that disproportionally impact those living in more deprived communities, which are exacerbated by health care disruptions like those caused by the SARS-CoV-2 pandemic.

A cytosine analog that confers enhanced potency to antisense oligonucleotides.

Antisense technology is based on the ability to design potent, sequence-specific inhibitors. The G-clamp heterocycle modification, a cytosine analog that clamps on to guanine by forming an additional hydrogen bond, was rationally designed to enhance oligonucleotide/RNA hybrid affinity. A single, context-dependent substitution of a G-clamp heterocycle into a 15-mer phosphorothioate oligodeoxynucleotide (S-ON) targeting the cyclin-dependent kinase inhibitor, p27(kip1), enhanced antisense activity as compared with a previously optimized C5-propynyl-modified p27(kip1) S-ON and functionally replaced 11 C5-propynyl modifications. Dose-dependent, sequence-specific antisense inhibition was observed at nanomolar concentrations of the G-clamp S-ONs. A single nucleotide mismatch between the G-clamp S-ON and the p27(kip1) mRNA reduced the potency of the antisense ON by five-fold. A 2-base-mismatch S-ON eliminated antisense activity, confirming the sequence specificity of G-clamp-modified S-ONs. The G-clamp-substituted p27(kip1) S-ON activated RNase H-mediated cleavage and demonstrated increased in vitro binding affinity for its RNA target compared with conventional 15-mer S-ONs. Furthermore, incorporation of a single G-clamp modification into a previously optimized 20-mer phosphorothioate antisense S-ON targeting c-raf increased the potency of the S-ON 25-fold. The G-clamp heterocycle is a potent, mismatch-sensitive, automated synthesizer-compatible antisense S-ON modification that will have important applications in the elucidation of gene function, the validation of gene targets, and the development of more potent antisense-based pharmaceuticals.

Cellular penetration and antisense activity by a phenoxazine-substituted heptanucleotide.

One of the major barriers to the development of antisense therapeutics has been their poor bioavailability. Numerous oligonucleotide modifications have been synthesized and evaluated for enhanced cellular permeation with limited success. Phenoxazine, a tricyclic 2' deoxycytidine analog, was designed to improve stacking interactions between heterocycles of oligonucleotide/RNA hybrids and to enhance cellular uptake. However, the bioactivity and cellular permeation properties of phenoxazine-modified oligonucleotides were unknown. Incorporation of four phenoxazine bases into a previously optimized C-5 propyne pyrimidine modified 7-mer phosphorothioate oligonucleotide targeting SV40 large T antigen enhanced in vitro binding affinity for its RNA target and redirected RNAse H-mediated cleavage as compared with the 7-mer C-5 propynyl phosphorothioate oligonucleotide (S-ON). The phenoxazine/C-5 propynyl U 7-mer S-ON showed dose-dependent, sequence-specific, and target-selective antisense activity following microinjection into cells. Incubation of the phenoxazine/C-5 propynyl U S-ON with a variety of tissue culture cells, in the absence of any cationic lipid, revealed unaided cellular penetration, nuclear accumulation, and subsequent antisense activity. The unique permeation properties and gene-specific antisense activity of the 7-mer phenoxazine/C-5 propynyl U S-ON paves the way for developing potent, cost-effective, self-permeable antisense therapeutics.

Potent and selective gene inhibition using antisense oligodeoxynucleotides.

The development of antisense technology as a generally useful tool relies on the use of potent agents and the utilization of many controls in experiments. Here we describe our experience using oligodeoxynucleotides (ODNs) containing C-5 propynyl pyrimidine and phosphorothioate modifications as broadly applicable gene inhibition agents in cell culture. Methods include selection of antisense sequences, synthesis and purification of ODNs, choice of controls, delivery methods (microinjection, cationic lipid transfection, and electroporation), and analysis of gene inhibition.

Structure-activity relationships in cell culture.

The use of antisense oligonucleotides in cell culture relies on the development of potent modifications and cell delivery techniques. C-5 propyne pyrimidine phosphorothioate oligonucleotides bind selectively and with high affinity to RNA within cells, leading to potent antisense inhibition. The effect that increasing steric bulk of C-5-substituted deoxyuridine analogues has on the affinity for RNA and the ability to inhibit gene expression is discussed. The GS 2888 cytofectin agent delivers oligonucleotides to cells at high efficiency in the presence of serum in cell media. Modifications leading to the discovery of GS 2888 centered on the aliphatic chain length of the molecule and the pKa of the polar head group. Together, the C-5 propyne modifications and the GS 2888 cytofectin agent have been shown to be effective inhibitors of gene expression in cell culture, particularly in the area of cell cycle proteins involved in cancer progression.

Antisense technology and prospects for therapy of viral infections and cancer.

Eighteen years ago, antisense oligonucleotide therapeutics that can selectively knock out disease-causing genes could easily have been viewed as science fiction. Yet today, through much persistence and focused investment, the technology has nearly evolved to the point of realization. A number of first-generation antisense compounds have entered human clinical trials. Some of these compounds appear to work by an antisense mechanism to inhibit the expression of disease-causing genes, while others probably work by unanticipated, yet clinically beneficial, mechanisms. In this review, the current status of antisense oligonucleotide development will be described as it relates to two areas of concentrated effort: antiviral and anticancer applications.

Antisense gene inhibition by C-5-substituted deoxyuridine-containing oligodeoxynucleotides.

Antisense oligodeoxynucleotides (ODNs) are capable of inhibiting gene expression via a RNase H mechanism in which the complementary RNA is degraded by RNase H. C-5 propyne dU phosphorothioate ODNs bind selectively and with high affinity to RNA within cells leading to potent antisense inhibition of RNA translation. The effect that increasing steric bulk of C-5-substituted deoxyuridine analogs has on affinity for RNA and ability to inhibit gene expression is discussed. The relative binding affinity was measured by thermal denaturation (Tm) analysis, and antisense activity was determined by inhibition of SV40 T-antigen (TAg) expression in CV1 cells. The results show that antisense activity is not directly correlated to Tm measurements. In vitro analysis (RNase H cleavage, on-rates, and off-rates) and pre-formed ODN/RNA experiments indicate that RNase H activity and intracellular dissociation appear to be major determinants of the antisense potency of the various substituted ODNs. The results of our analysis point to the unique ability of C-5 propyne dU ODNs to selectively bind to RNA within cells and activate cleavage of RNA by RNase H leading to potent inhibition of gene expression.

In pursuit of antisense.

The first generation of antisense oligodeoxynucleotides (ODNs) are now undergoing clinical trials, but their effects may reflect biological activities unrelated to their ability to bind RNA. Nevertheless, preclinical animal studies now suggest that phosphorothioate ODNs may be more permeable in certain animal tissues than in cell culture, raising hopes that antisense mechanisms can be exploited pharmacologically.

Elucidation of gene function using C-5 propyne antisense oligonucleotides.

Identification of human disease-causing genes continues to be an intense area of research. While cloning of genes may lead to diagnostic tests, development of a cure requires an understanding of the gene's function in both normal and diseased cells. Thus, there exists a need for a reproducible and simple method to elucidate gene function. We evaluate C-5 propyne pyrimidine modified phosphorothioate antisense oligonucleotides (ONs) targeted against two human cell cycle proteins that are aberrantly expressed in breast cancer: p34cdc2 kinase and cyclin B1. Dose-dependent, sequence-specific, and gene-specific inhibition of both proteins was achieved at nanomolar concentrations of ONs in normal and breast cancer cells. Precise binding of the antisense ONs to their target RNA was absolutely required for antisense activity. Four or six base-mismatched ONs eliminated antisense activity confirming the sequence specificity of the antisense ONs. Antisense inhibition of p34cdc2 kinase resulted in a significant accumulation of cells in the Gap2/mitosis phase of the cell cycle in normal cells, but caused little effect on cell cycle progression in breast cancer cells. These data demonstrate the potency, specificity, and utility of C-5 propyne modified antisense ONs as biological tools and illustrate the redundancy of cell cycle protein function that can occur in cancer cells.

Nucleosides with a twist. Can fixed forms of sugar ring pucker influence biological activity in nucleosides and oligonucleotides?

The sugar moiety of nucleosides in solution is known to exist in a rapid dynamic equilibrium between extreme Northern and Southern conformations as defined in the pseudorotational cycle. In the present work, we describe how the bicyclo[3.1.0]hexane template fixes the ring pucker of 2'-deoxy-methanocarba-nucleosides 1-5 and 12 to values corresponding to either one of these two extreme conformations that are typical of nucleosides. The syntheses of the fixed Northern conformers 1-5 were performed by Mitsunobu coupling of the heterocyclic bases with the chiral carbocyclic alcohol 6 [(1R,2S,4R,5S)-1-[(benzyloxy)methyl]-2-(tert-butyloxy)-4-hydrox ybicyclo[3.1.0]hexane], while the synthesis of the Southern conformer, (S)-methanocarba-T (12), was reported earlier. Carbocyclic thymidine (carba-T, 13) was used as a reference, flexible carbocyclic nucleoside. Antiviral evaluation of these compounds revealed a very potent antiherpetic activity associated with the Northern thymidine analogue 2, which was more powerful than the reference standard acyclovir against both HSV-1 and HSV-2. (N)-Methanocarba-T (2) was further evaluated as a component of a short oligodeoxynucleotide (ODN) phosphorothioate (5'-CTTCATTTTTTCTTC-3') where all thymidines were replaced by 2. The expected thermodynamic stability resulting from the preorganization of the pseudosugar rings into a Northern conformation, typical of A-DNA, was evident by the increase in Tm of the corresponding DNA/RNA heteroduplex. However, the rigid A-tract ODN caused loss of RNase H recruitment. A detailed conformational analysis of (N)-methanocarba-T (2) and (S)-methanocarba-T (12), as representative examples of conformationally rigid pseudorotational antipodes, revealed that in addition to their different forms of ring pucker, (S)-methanocarba-T appears to be a rather stiff molecule with fewer low-energy conformational states available compared to (N)-methanocarba-T. The syn/anti-energy barrier for these nucleoside analogues is 5-6 kcal/mol higher than for common nucleosides.

Effects of oligonucleotide length, mismatches and mRNA levels on C-5 propyne-modified antisense potency.

To understand the parameters required for designing potent and specific antisense C-5 propynyl-pyrimidine-2'-deoxyphosphorothioate-modified oligonucleotides (C-5 propyne ONs), we have utilized a HeLa line that stably expresses luciferase under tight control of a tetracycline-responsive promoter. Using this sensitive and regulatable cell-based system we have identified five distinct antisense ONs targeting luciferase and have investigated the role that ON length, target mismatches, compound stability and intracellular RNA levels play in affecting antisense potency. We demonstrate that C-5 propyne ONs as short as 11 bases retained 66% of the potency demonstrated by the parent 15 base compound, that a one base internal mismatch between the antisense ON and the luciferase target reduced the potency of the antisense ON by 43% and two or more mismatches completely inactivated the antisense ON and that C-5 propyne ONs have a biologically active half-life in tissue culture of 35 h. In addition, by regulating the intracellular levels of the luciferase mRNA over 20-fold, we show that the potency of C-5 propyne ONs is unaffected by changes in the expression level of the target RNA. These data suggest that low and high copy messages can be targeted with equivalent potency using C-5 propyne ONs.

Oligodeoxynucleotides containing C-7 propyne analogs of 7-deaza-2'-deoxyguanosine and 7-deaza-2'-deoxyadenosine.

The synthesis, hybridization properties and antisense activities of oligodeoxynucleotides (ODNs) containing 7-(1-propynyl)-7-deaza-2'-deoxyguanosine (pdG) and 7-(1-propynyl)-7-deaza-2'-deoxyadenosine (pdA) are described. The suitably protected nucleosides were synthesized and incorporated into ODNs. Thermal denaturation (Tm) of these ODNs hybridized to RNA demonstrates an increased stability relative to 7-unsubstituted deazapurine and unmodified ODN controls. Antisense inhibition by these ODNs was determined in a controlled microinjection assay and the results demonstrate that an ODN containing pdG is approximately 6 times more active than the unmodified ODN. 7-Propyne-7-deaza-2'-deoxyguanosine is a promising lead analog for the development of antisense ODNs with increased potency.

Potent and selective inhibition of gene expression by an antisense heptanucleotide.

Factors that govern the specificity of an antisense oligonucleotide (ON) for its target RNA include accessibility of the targeted RNA to ON binding, stability of ON/RNA complexes in cells, and susceptibility of the ON/RNA complex to RNase H cleavage. ON specificity is generally proposed to be dependent on its length. To date, virtually all previous antisense experiments have used 12-25 nt-long ONs. We explored the antisense activity and specificity of short (7 and 8 nt) ONs modified with C-5 propyne pyrimidines and phosphorothioate internucleotide linkages. Gene-selective, mismatch sensitive, and RNase H-dependent inhibition was observed for a heptanucleotide ON. We demonstrated that the flanking sequences of the target RNA are a major determinant of specificity. The use of shorter ONs as antisense agents has the distinct advantage of simplified synthesis. These results may lead to a general, cost-effective solution to the development of antisense ONs as therapeutic agents.

A serum-resistant cytofectin for cellular delivery of antisense oligodeoxynucleotides and plasmid DNA.

Development of antisense technology has focused in part on creating improved methods for delivering oligodeoxynucleotides (ODNs) to cells. In this report, we describe a cationic lipid that, when formulated with the fusogenic lipid dioleoylphosphatidyliethanolamine, greatly improves the cellular uptake properties of antisense ODNs, as well as plasmid DNA. This lipid formulation, termed GS 2888 cytofectin, (i) efficiently transfects ODNs and plasmids into many cell types in the presence or absence of 10% serum in the medium, (ii) uses a 4- to 10-fold lower concentration of the agent as compared to the commercially available Lipofectin liposome, and (iii) is > or = 20-fold more effective at eliciting antisense effects in the presence of serum when compared to Lipofectin. Here we show antisense effects using GS 2888 cytofectin together with C-5 propynyl pyrimidine phosphorothioate ODNs in which we achieve inhibition of gene expression using low nanomolar concentrations of ODN. This agent expands the utility of antisense ODNs for their use in understanding gene function and offers the potential for its use in DNA delivery applications in vivo.

Site and mechanism of antisense inhibition by C-5 propyne oligonucleotides.

Antisense gene inhibition occurs when an oligonucleotide (ON) has sufficient binding affinity such that it hybridizes its reverse complementary target RNA and prevents translation either by causing inactivation of the RNA (possibly by RNase H) or by interfering with a cellular process such as stalling a ribosome. The mechanisms underlying these processes were explored. Cellular antisense inhibition was evaluated in a microinjection assay using ON modifications which precluded or allowed in vitro RNase H cleavage of ON/RNA hybrids. RNase H-independent inhibition of protein synthesis could be achieved by targeting either the 5'-untranslated region or the 5'-splice junction of SV40 large T antigen using 2'-O-allyl phosphodiester ONs which contained C-5 propynylpyrimidines (C-5 propyne). Inhibition at both sites was 20-fold less active than inhibition using RNase H-competent C-5 propyne 2'-deoxy phosphorothioate ONs. In vitro analysis of association and dissociation of the two classes of ONs with complementary RNA showed that the C-5 propyne 2'-O-allyl phosphodiester ON bound to RNA as well as the C-5 propyne 2'-deoxy phosphorothioate ON. In vitro translation assays suggested that the two classes of ONs should yield equivalent antisense effects in the absence of RNase H. Next, ON/T antigen RNA hybrids were injected into the nuclei and cytoplasm of cells. Injection of C-5 propyne 2'-O-allyl phosphodiester ON/RNA hybrids resulted in expression of T antigen, implying that the ONs dissociated from the RNA in cells which likely accounted for their low potency. In contrast, when C-5 propyne 2'-deoxy phosphorothioate ON/T antigen RNA complexes were injected into the nucleus, the duplexes were stable enough to completely block T antigen translation, presumably by RNA inactivation. Thus, a dramatic finding is that C-5 propyne 2'-deoxy phosphorothioate ONs, once hybridized to RNA, are completely effective at preventing mRNA translation. The implication is that further increases in complex stability coupled with effective RNase H cleavage will not result in enhanced potency. We predict that the development of more effective ONs will only come from modifications which increase the rate of ON/RNA complex formation within the nucleus.

Gene inhibition using antisense oligodeoxynucleotides.

Antisense oligodeoxynucleotides (ODNs) have great promise as agents for the specific manipulation of gene expression. Until recently, nonspecific effects of ODNs often confounded the interpretation of antisense studies. Improvements in ODN chemistry and cellular delivery techniques now allow for more potent and specific gene inhibition. This review critically evaluates recent progress in the development of antisense ODNs.