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Hemophilia B is an X-chromosome-linked recessive disorder that is caused by a deficiency of biologically active clotting factor IX (FIX). In this work, liposomes (Lip) were used for non-viral, in vivo gene transfer of the human FIX gene into mouse organs. Plasmid DNA, containing the human FIX cDNA under the control of the Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR), was encapsulated in 1-2-microns multilamellar Lip composed of egg phosphatidylcholine (EPC). The percentage of Lip-associated DNA was 47%, and 72% of the Lip DNA was protected from DNase I digestion. The Lip-encapsulated (Len) DNA was injected intravenously into Balb/c mice, and at various times post-injection, various tissues were examined for the presence of the exogenous DNA. Plasmid DNA was detected by Southern blot analysis mainly in the liver and spleen, but small amounts were also detected in the lungs, heart and kidneys. The plasmid DNA was retained in mouse liver cells for at least 7 days post-injection, and remained in an episomal state. The levels of human FIX protein in the mouse plasma were 190-650 pg per ml for 2 to 7 days post-injection. Treatment of mice with chloroquine (Cq) and colchicine (Cc) prior to Lip injection significantly increased the amount of plasmid DNA found in the liver cells, as well as the level of human FIX in the plasma. These results demonstrate the potential use of Len DNA for gene transfer into liver and spleen, and for gene therapy of inherited and acquired disorders.

Human papillomavirus (HPV) was detected by using two sets of deoxyribonucleotide primers for differentiating between 'low-risk' types (HPV11 and HPV6) and 'high-risk' (hri) types (HPV16, HPV18 and HPV33). A new application of the Chemiprobe method for labeling DNA was used to detect products of the polymerase chain reaction (PCR) from 36 cervical biopsies. This method, first demonstrated by Uchimura et al. (submitted), is based on the sulfonation of a polycytidylic acid tail of 5-20 monomers attached to the 5' end of either one or both of the PCR primers. This procedure can increase the sensitivity of detection of PCR products more than 100-fold with respect to ethidium bromide (EtdBr) staining. Various methods were used to detect hri HPV DNA in the 36 clinical samples. The number of positive results obtained was as follows, two by Southern-blot hybridization; five by PCR amplification followed by electrophoresis and detection of products by EtdBr staining; six by PCR amplification using one or two sulfonated C-tailed primers followed by electroblotting and immunoenzymatic visualization; and five by hybridization of sulfonated genomic viral recombinant with a PCR product immobilized on a membrane. The yield of the PCR product was significantly greater when one of the primers was C-tailed than when both or neither of the primers were C-tailed. PCR employing sulfonated C-tailed oligo primers is very specific and sensitive, and the entire procedure can be employed as a nonradioactive substitute for radioactive dot-blot or Southern-blot hybridization procedures, routinely used for detection of HPV in clinical samples.