Gene therapy offers a radical different approach to the treatment of arthritis. Here we have demonstrated that two marker genes (lacZ and neo) and cDNA coding for a potentially therapeutic protein (human interleukin 1-receptor-antagonist protein; IRAP or IL-lra) can be delivered, by ex vivo techniques, to the synovial lining of joints; intraarticular expression of IRAP inhibited intraarticular responses to interleukin 1. To achieve this, lapine synoviocytes were first transduced in culture by retroviral infection. The genetically modified synovial cells were then transplanted by intraarticular I jection into the knee joints of rabbits, where they efficiently colonized the synovium. Assay of joint lavages confirmed the in vivo expression of biologically active human IRAP. With allografted cells, IRAP expression was lost by 12 days after transfer. In contrast, autografted synoviocytes continued to express IRAP for -5 weeks. Knee joints expressing human IRAP were protected from the leukocytosis that otherwise follows the intraarticular injection of recombinant human interleukin 1p3. Thus, we report the intraarticular expression and activity of a potentially therapeutic protein by genetransfer technology; these experiments demonstrate the feasibility of treating arthritis and other joint disorders with gene therapy.Arthritis is a chronic, debilitating condition affecting over 30 million Americans (1). Presently incurable, it remains the agent of considerable suffering and economic loss. Therapeutic intervention in arthritis is hindered by a number of factors, including difficulties in targeting drugs to joints. Proteins are particularly vulnerable to this restriction, which is of special concern, as many new agents with considerable antiarthritic potential are proteins. As an alternative to traditional methods of drug delivery, we have suggested the transfer oftherapeutic genes to the synovial lining ofjoints (2,3). Expression of these genes would overcome proteindelivery problems and lead to the intraarticular accumulation of the gene products at the site of disease, with reduced exposure of nontarget organs.Using the rabbit knee joint as a model system, we are therefore developing in vivo and ex vivo methods for delivering genes to joints. This model takes advantage of the similarity in size between the knee joint of the rabbit and the human proximal interphalangeal joint, a common site of rheumatoid arthritis. Moreover, there exists a large body of literature on the biology of the rabbit's knee, including well-established methods for synovial cell culture (e.g., refs. 4-6). Here we report the transfer to synovium of two marker genes and one potentially therapeutic gene by an ex vivo approach. Intraarticular expression of the interleukin 1-re-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. (rhIL-1,8). These results demonstrate the feasibility of ...
Macrophage inflammatory protein-2 (MIP-2) is a member of a family of cytokines that play roles in inflammatory, immune, and wound healing responses. To clone the cDNA for rat MIP-2, RNA was isolated from the lungs of Fischer 344 rats after instillation of lipopolysaccharide. Reverse transcription-polymerase chain reaction was performed by using synthetic oligonucleotide primers designed from the mouse MIP-2 cDNA sequence. A cDNA containing the coding region of rat MIP-2 was cloned and sequenced. Comparison to the mouse MIP-2 cDNA demonstrated 90.3% homology at the nucleotide level and 86% homology at the amino acid level. The rat MIP-2 cDNA was expressed in Escherichia coli and protein evaluated for bioactivity. The recombinant rat MIP-2 was chemotactic for rat neutrophils but did not stimulate migration of rat alveolar macrophages or human peripheral blood eosinophils or lymphocytes. In addition, the recombinant rat MIP-2 and the related rat chemokine, KC/CINC stimulated proliferation of rat alveolar epithelial cells but not fibroblasts in vitro.
Gyrate atrophy of the choroid and retina (GA) is an autosomal recessive chorioretinal degeneration caused by deficiency of the mitochondrial matrix enzyme, ornithine-5-aminotransferase (OAT). To study the molecular basis of the mutations causing GA, we cloned and sequenced the human OAT cDNA and determined the intron-exon arrangement of the structural gene. Using the cDNA template, we synthesized antisense RNA probes and performed RNase A protection experiments with RNA from four Lebanese GA patients. We found a probe-target mismatch at the 5' end of the first coding exon and amplified this region of the patients' genomic DNA using the polymerase chain reaction. Sequence analysis showed a G A transition, changing the initiator ATG (methionine) codon to ATA. This mutation segregates with the GA allele in both pedigrees. Initiation of translation at the closest in-frame methionine codon would truncate OAT by 138 amino acids, eliminating the entire mitochondrial leader sequence and 113 amino acids of the mature peptide.
IVD i s a m i t o c h o n d r i a l f l a v o p r o t e i n , a tetramer o f 43 kDasubunits, which c a t a l y z e s t h e t h i r d r e a c t i o n i n l e u c i n e metabol i s m . H e r e d i t a r y d e f i c i e n c y o f IVO causes i s o v a l e r i c acidemia, one o f t h e more common i n h e r i t e d o r g a n i c acidemias, c h a r a c t e r i zed by r e c u r r e n t episodes o f vomiting, ketoacidosis, l e t h a r g y and sweaty f e e t odor. Extensive molecular heterogeneity, i n c l u d i n g a t l e a s t f i v e d i s t i n c t v a r i a n t a l l e l e s , has been shown on t h e b a s i s o f t h e presence o r absence o f immunoprecipitable v a r i a n t IVDs and t h e i r s i z e s (PNAS 82:7081, 1985). As a f i r s t s t e p i n t h e study o f t h e molecular b a s i s o f i s o v a l e r i c acidemia, we have i s o l a t e d a cDNA clone encoding r a t IVD. F i r s t , we h i g h l y p u r if i e d t h e mRNA encoding IVD from r a t l i v e r by polysome immunop u r i f i c a t i o n u s i n g p o l y c l o n a l , monospecific antibody. A cDNA l i b r a r y , enriched f o r IVD, was prepared u s i n g t h e p u r i f i e d mRNA, and screened by c o l o n l y h y b r i d i z a t i o n u s i n g two o l i g o n u c l e o t i d e s (17-mer and 15-mer) corresponding t o t h e p o r t i o n s o f t h e NHt e r m i n a l amino a c i d sequence.Numerous p o s i t i v e cDNA c l on& were i s o l a t e d . One o f them contained a 54 bp p o r t i o n which perf e c t l y matched an 18 amino a c i d p e p t i d e from t h e amino terminus o f pure r a t IVO, thereby p o s i t i v e l y i d e n t i f y i n g t h e c l o n e as t h a t o f r a t IVD. T h i s cDNA extends 100 bp towards t h e 5 ' end, i n d i c a t i n g t h a t i t c o n t a i n s t h e e n t i r e sequence f o r t h e l e a d e r p e p t i d e ( 2 kDa). Screening o f human cDNA l i b r a r i e s f o r IVD cDNA r c i n n m n m c c Second trimester MSAFP screening for low values is proving valuable in the detection of fetal chromosome defects in women 4 3 5 yrs. of age. Detection prior to 16 wks. would be desirable. To determine whether low MSAFP values also occur in the first trimester in association with a fetal chromosomal abnormality, we used a "simultaneous-sandwich" radioimmunoassay with polystyrene beads coated with anti-AFP monoclonal antibodies, using solid phase support. The coefficient of variation was45% for this assay which is about 10 x more sensitive than conventional RIAs .Blood was sampled in 359 cases just before CVS (chorionic villus sampling) in Milan. Fetal age was usually assessed by 2 ultrasound studies' 1 wk. apart, and chromosomes of the villi analysed. Sera were sent blind & only after AFP assay results were sent to Milan were karyotypes released. MSAFPL0. Univ o f Toronto, Ontario. MCAD i s a mitochondria1 f l a v o p r o t e i n , a tetramer o f 45 kDa OAT i s a homotetrameric, mitochondria1 m a t r i x enzyme d e f isubunits, which c a t a l y z e s t h e f i r s t r e a c t i o n i n t h e b -o x i d a t...
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