Progress in the delivery of therapeutic oligonucleotides: organ/cellular distribution and targeted delivery of oligonucleotides in vivo.

Oligonucleotide (ODN) therapy is a powerful tool for modulation of gene expression in vivo. With advances in ODN chemistry and progress in formulation development, ODNs are becoming widely acceptable drugs. This review summarizes the current status and future trend of the in vivo application of ODN therapeutics, especially antisense ODNs. Here, we review the current understanding of the tissue/organ distribution and cellular uptake of ODN drugs administered parenterally or nonparenterally to intact animals. The problems and advantages inherent in the use of different delivery methods for the treatment of particular diseases are discussed in detail. Emphasis is placed on the most widely studied ODN analogs, the phosphorothioates (PS). Lessons learned from antisense PS studies have broad implications for ODN therapeutics in general.

THE EVOLUTIONARY GENETIC STATUS OF ICELANDIC EELS.

The Iceland population of Anguilla eels contains an elevated frequency of fish with vertebral numbers lower than those typical of European localities. Several distinct hypotheses have been advanced to account for these morphologically atypical fish: for example, they could represent (1) genetically "pure" American expatriates, (2) genetically "pure" European types with ontogenetic abnormalities, or (3) hybrids between American and European forms. Here we critically test these and other possibilities by examining the joint distributions of allozyme markers, mitochondrial DNA markers, and vertebral numbers in Icelandic eels. The particular patterns of association among the genetic and morphological traits demonstrate that the Iceland population includes, in low frequency, the products of hybridization between American and European eels. Approximately 2-4% of the gene pool in the Iceland eel population is derived from American eel ancestry. This hybrid zone is highly unusual in the biological world, because the mating events in catadromous eels presumably take place thousands of kilometers from where the hybrids are observed as maturing juveniles. The molecular data, in conjunction with the geographic distributions, strongly suggest that the differences in migrational behavior and morphology between American and European eels include an important additive genetic component. Evolutionary hypotheses are advanced to account for the original separation of North Atlantic eels into American and European populations, and for the presence of hybrids in Iceland.

EXCLUSION OF THE ROLE OF SECONDARY CONTACT IN AN ALLELE FREQUENCY CLINE IN THE MUSSEL MYTILUS EDULIS.

We examined the hypothesis that secondary contact generates an allele-frequency cline at the aminopeptidase-I locus (Lap) in the marine mussel, Mytilus edulis. It has been proposed that variation at the Lap locus is neutral and that the cline results from secondary contact between differentiated oceanic and estuarine populations (Levinton, 1980). We tested this hypothesis by comparing the genotypic distributions in samples from the cline to distributions that incorporate mixing effects. We employed a statistical model that determines the degree of contact using a maximum likelihood estimator and then incorporates the mixing estimates into an expected distribution of genotypes. Wahlund effects resulting from possible admixture are thereby incorporated into the expected distribution. Failure of the model to reconcile the observed with the expected distribution of genotypes indicates that the observed population structure does not result from admixture. The null hypothesis of mixing was unable to explain about 33% of the samples. Combined tests demonstrated the general departure from the mixing model to be highly significant. The distribution of heterozygote discrepancies across the cline was inconsistent with the expectations of a mixing model. Therefore we reject explanations for the structure of the Lap cline that involve secondary contact. Selection directed at the Lap locus appears necessary to explain the genotypic structure of clinal populations.

THE PHYSIOLOGICAL BASIS OF NATURAL SELECTION AT THE LAP LOCUS.

An extensive research program was undertaken to evaluate the contribution of genetic variation at the Lap locus to variation in physiological traits under natural conditions. Rates of carbon and nitrogen metabolism were monitored in a population of the mussel Mytilus edulis near the center of the Lap allele frequency cline on the north shore of Long Island. The goal of this research was to establish whether the previously described genotype-dependent differences in physiological phenotype are meaningful in ecologically relevant circumstances. It was predicted from laboratory studies that, in nature, genotype-dependent differences will exist for rates of nitrogen excretion and that other aspects of the animal's physiology, particularly rates of carbon metabolism, will be unaffected by Lap genotype. Rates of amino acid and ammonia excretion were significantly dependent upon Lap genotype; individuals with the Lap94 allele exhibited greater rates of nitrogen loss. These differences among genotypes were most evident in the fall, between September and December. The genotype-dependent component of rates of nitrogen loss were also largest relative to the total rate of excretion during the fall period. As predicted, other aspects of the nitrogen metabolism (acquisition) and rates of carbon metabolism were independent of Lap genotype. There was a striking congruity among a variety of observations that all indicate that phenotypic differences in nitrogen metabolism are the basis of natural selection at the Lap locus in Long Island Sound. Rates of growth were minimal during the fall months (Hilbish, 1985) and mussels are known to lose weight in a genotype-specific manner during this period (Koehn et al., 1980). Rates of elemental gain and loss were summed to produce carbon and nitrogen budgets; these data show the fall to be a period of extended deficit in carbon and nitrogen balance. Genotype-dependent losses of ammonia and amino acids were greatest during the fall months. Finally, selection against the Lap94 allele occurs predominantly in the fall (Hilbish, 1985). The data indicate that the depletion of nitrogen resources provides the basis for selection against Lap94 genotypes during the fall months.