Nucleic acid‐based therapeutics that regulate gene expression have been developed towards clinical use at a steady pace for several decades, but in recent years the field has been accelerating. To date, there are 11 marketed products based on antisense oligonucleotides, aptamers and small interfering RNAs, and many others are in the pipeline for both academia and industry. A major technology trigger for this development has been progress in oligonucleotide chemistry to improve the drug properties and reduce cost of goods, but the main hurdle for the application to a wider range of disorders is delivery to target tissues. The adoption of delivery technologies, such as conjugates or nanoparticles, has been a game changer for many therapeutic indications, but many others are still awaiting their eureka moment. Here, we cover the variety of methods developed to deliver nucleic acid‐based therapeutics across biological barriers and the model systems used to test them. We discuss important safety considerations and regulatory requirements for synthetic oligonucleotide chemistries and the hurdles for translating laboratory breakthroughs to the clinic. Recent advances in the delivery of nucleic acid‐based therapeutics and in the development of model systems, as well as safety considerations and regulatory requirements for synthetic oligonucleotide chemistries are discussed in this review on oligonucleotide‐based therapeutics.

Microcell hybrid lines of A9 mouse fibrosarcoma containing complete or partially deleted human chromosomes 3 (chr. 3) were inoculated into SCID mice. Cell lines derived from the tumors were examined by fluorescent in situ hybridization for the status of the transferred human chromosome and by PCR for marker loss. The SCID tumors arising after the inoculation of 10(5) cells were passaged serially in vivo and regularly showed loss of four markers; D3S1029 (3p21.3-21.2), AP20R (3p22-21.3, D3S32 (3p21.3-p21.2), and THRB (3p24). This regularly deleted region is bordered by markers GNA12 (3p21.1-p21.3) and VHL (3p25) that were maintained in a fraction of tumors. Fragments derived from the long arm of chromosome 3 and corresponding markers in the 3q26-q28 region were retained in all tumors. Our findings may be related to the postulated presence of tumor suppressor genes in the 3p24-p21 region as indicated by the frequent deletion of this region in renal and small cell lung carcinomas and other solid tumors. The technically cumbersome identification of suppressor genes may be supplemented by an "elimination test" based on analogous principles.

We have applied a functional test for tumour antagonizing genes based on human chromosome 3 (chr3)–mouse fibrosarcoma A9 MCHs that were studied in vitro and after growth as tumours in severe combined immunodeficiency (SCID) mice. Previously, we reported that 9 out of the 36 SCID‐tumours maintained the transferred chr3 (“chr3+” tumours), but lost the expression of the known human TSG fragile histidine triad gene (FHIT) in contrast to 14 other 3p‐genes examined. Here we report the results of the duplex RT‐PCR analysis of 9 “chr3+” tumours and 3 parental MCHs. We have examined the expression of 34 human 3p‐genes from known cancer‐related regions of instability, including 13 genes from CER1 defined by us previously at 3p21.33–p21.31 and 10 genes from the LUCA region at 3p21.31. We have found that in addition to FHIT, expression of the LTF gene from CER1 at 3p21.33‐p21.31 was lost in all 9 tumours analyzed. The transcript of the solute carrier family 38 member 3 gene (SLC38A3) gene from LUCA region at 3p21.31 was not found in 8 and was greatly reduced in 1 out of these 9 tumours. Expression of the down‐regulated in renal cell carcinoma gene (DRR1) gene at 3p14.2 was lost in 7 and down regulated in 2 “chr3+” tumours. In the SCID‐tumour derived cell lines treatment with 5‐aza‐2′‐deoxycytidine restored the mRNA expression of LTF, indicating the integrity of DNA sequences. Notably that transcription of the LTF and 2 flanking genes, LRRC2 and TMEM7, as well as transcription of the SLC38A3 gene, were also impaired in all 5 RCC cell lines analyzed. Our data indicate these genes as putative tumour suppressor genes. © 2006 Wiley‐Liss, Inc.

By passaging microcell hybrids (MCHs) containing human chromosome 3 (chr3) on A9 mouse fibrosarcoma background through severe combined immunodeficient (SCID) mice (elimination test), we have previously defined a 1-Mb-long common eliminated region 1 (CER1) at 3p21.3, a second eliminated region (ER2) at 3p21.1-p14 and a common retained region (CRR) at 3q26-qter. In the present work, chr3 was transferred by microcell fusion into the human nonpapillary renal cell carcinoma line KH39 that contained uniparentally disomic chr3. Four MCHs were generated. Compared with KH39, they developed fewer and smaller tumors, which grew after longer latency periods in SCID mice. The tumors were analyzed in comparison with corresponding MCHs by chr3 arm-specific painting, 19 fluorescent in situ hybridization (FISH) probes, and 27 polymorphic markers. Three MCHs that maintained the intact exogenous chr3 in vitro lost one 3p copy in all 11 tumors. Seven of 11 tumors lost the exogenous 3p, whereas four tumors contained mixed cell populations that lacked either the exogenous or one endogenous KH39 derived 3p. In one MCH the exogenous chr3 showed deletions within CER1 and ER2 already in vitro. It remained essentially unchanged in 8͞9 derived tumors. The third, exogenous copy of the 3q26 -q27 region (part of CRR) was retained in 16͞20 tumors. It can be concluded that the human͞human MCH-based elimination test identifies similar eliminated and retained regions on chr3 as the human͞murine MCH-based test.

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