Grafting genetically modified cells to the damaged brain: restorative effects of NGF expression.

Fibroblasts were genetically modified to secrete nerve growth factor (NGF) by infection with a retroviral vector and then implanted into the brains of rats that had surgical lesions of the fimbria-fornix. The grafted cells survived and produced sufficient NGF to prevent the degeneration of cholinergic neurons that would die without treatment. In addition, the protected cholinergic cells sprouted axons that projected in the direction of the cellular source of NGF. These results indicate that a combination of gene transfer and intracerebral grafting may provide an effective treatment for some disorders of the central nervous system.

Direct gene transfer into mouse muscle in vivo.

RNA and DNA expression vectors containing genes for chloramphenicol acetyltransferase, luciferase, and beta-galactosidase were separately injected into mouse skeletal muscle in vivo. Protein expression was readily detected in all cases, and no special delivery system was required for these effects. The extent of expression from both the RNA and DNA constructs was comparable to that obtained from fibroblasts transfected in vitro under optimal conditions. In situ cytochemical staining for beta-galactosidase activity was localized to muscle cells following injection of the beta-galactosidase DNA vector. After injection of the DNA luciferase expression vector, luciferase activity was present in the muscle for at least 2 months.

Clonal characteristics of experimentally induced "atherosclerotic" lesions in the hybrid hare.

The female hybrid hare (Lepus timidus x Lepus europaeus) is heterozygous for electrophoretically separable, X-linked isoenzymes of glucose-6-phosphate dehydrogenase. The isoenzymes of this animal have been used as cellular markers in the study of the clonal origins of experimentally induced atherosclerotic lesions. Aortic lesions produced in the hybrid hare by feeding cholesterol and injuring the aortic wall with a catheter have been shown to have polyclonal characteristics and in this way are fundamentally different from atherosclerotic fibrous plaques in man.

Nutritional management of PKU with glycomacropeptide from cheese whey.

Individuals with phenylketonuria (PKU) must follow a lifelong low-phenylalanine (Phe) diet to prevent neurological impairment. Compliance with the low-Phe diet is often poor owing to restriction in natural foods and the requirement for consumption of a Phe-free amino acid formula or medical food. Glycomacropeptide (GMP), a natural protein produced during cheese-making, is uniquely suited to a low-Phe diet because when isolated from cheese whey it contains minimal Phe (2.5-5 mg Phe/g protein). This paper reviews progress in evaluating the safety, acceptability and efficacy of GMP in the nutritional management of PKU. A variety of foods and beverages can be made with GMP to improve the taste, variety and convenience of the PKU diet. Sensory studies in individuals with PKU demonstrate that GMP foods are acceptable alternatives to amino acid medical foods. Studies in the PKU mouse model demonstrate that GMP supplemented with limiting indispensable amino acids provides a nutritionally adequate source of protein and improves the metabolic phenotype by reducing concentrations of Phe in plasma and brain. A case report in an adult with classical PKU who followed the GMP diet for 10 weeks at home indicates safety, acceptability of GMP food products, a 13-14% reduction in blood Phe levels (p<0.05) and improved distribution of dietary protein throughout the day compared with the amino acid diet. In summary, food products made with GMP that is supplemented with limiting indispensable amino acids provide a palatable alternative source of protein that may improve dietary compliance and metabolic control of PKU.

pH-sensitive, cationic liposomes: a new synthetic virus-like vector.

We describe the use of cationic, pH-sensitive liposomes to mediate the efficient transfer of DNA into a variety of cells in culture. Cationic lipids, containing an amine with a pK within the physiologic range of 4.5 to 8, were synthesized and incorporated with dioleoylphosphatidylethanolamine into liposomes. Acid conditions promoted DNA-binding, DNA-incorporation, and DNA-induced fusion by these cationic, pH-sensitive liposomes. Transfection efficiency in cultured cells was dependent on endosomal acidification in a manner akin to acidic-induced endosomal release of viruses. These liposomes constitute a promising new class of reagents for gene therapy.

DNA vector chemistry: the covalent attachment of signal peptides to plasmid DNA.

The nuclear entry of exogenous DNA in mammalian cells is critical for efficient gene transfer. A novel technique was developed for the covalent attachment of cationic peptides to double-stranded DNA using a cyclo-propapyrroloindole cross-linker. The attachment of the SV40 large T antigen nuclear localization signal peptide induced the nuclear accumulation of the conjugated DNA in digitonin-permeabilized cells via the classical pathway for the nuclear transport of karyophilic proteins. Increased nuclear uptake of the modified DNA, however, did not occur after it was microinjected into the cytoplasm of cultured cells. This demonstration that the covalent modification of DNA with a signal peptide alters its behavior and interaction with other cellular factors portends the potential of DNA vector chemistry to enhance the efficiency of cellular gene transfer.

Postnatal gene transfer into the central nervous system.

Substantial progress has been made in the development of techniques for the expression of foreign genes in the central nervous system of postnatal animals. Fetal and adult brain cells and other cells, including fibroblasts and muscle cells, have been successfully employed as vehicles for foreign gene expression in the central nervous system. Direct gene transfer strategies, such as those using herpes and adenoviral vectors, are presently under intense and fruitful investigation.

Layer-by-layer deposition of oppositely charged polyelectrolytes on the surface of condensed DNA particles.

DNA can be condensed with an excess of poly-cations in aqueous solutions forming stable particles of submicron size with positive surface charge. This charge surplus can be used to deposit alternating layers of polyanions and polycations on the surface surrounding the core of condensed DNA. Using poly-L-lysine (PLL) and succinylated PLL (SPLL) as polycation and polyanion, respectively, we demonstrated layer-by-layer architecture of the particles. Polyanions with a shorter carboxyl/backbone distance tend to disassemble binary DNA/PLL complexes by displacing DNA while polyanions with a longer carboxyl/backbone distance effectively formed a tertiary complex. The zeta potential of such complexes became negative, indicating effective surface recharging. The charge stoichiometry of the DNA/PLL/SPLL complex was found to be close to 1:1:1, resembling poly-electrolyte complexes layered on macrosurfaces. Recharged particles containing condensed plasmid DNA may find applications as non-viral gene delivery vectors.

Complexes of non-cationic liposomes and histone H1 mediate efficient transfection of DNA without encapsulation.

Transfection competent complexes were assembled using a three component system. The constituents of the basic system were plasmid DNA, cationic DNA binding protein (NLS-H1) and anionic liposomes (dioleoyl phosphatidylethanolamine (DOPE) or phosphatidylserine (PS)). In contrast to cationic liposome/DNA binary complexes, all of the DNA in these ternary complexes was sensitive to DNase I degradation and ethidium bromide intercalation. Transmission electron microscopy revealed that these ternary complexes formed unique structures in which the DNA was located either on the outside of individual liposomes or bridging two or more liposomes. This provides evidence that plasmid DNA encapsulation is not essential for transfection competency.

An early history of gene transfer and therapy.

The term "gene therapy" was coined to distinguish it from the Orwellian connotations of "human genetic engineering," which, in turn, was derived from the term "genetic engineering." Genetic engineering was first used at the Sixth International Congress of Genetics held in 1932 and was taken to mean "the application of genetic principles to animal and plant breeding." Once the basics of molecular genetics and gene transfer in bacteria were established in the 1960s, gene transfer into animals and humans using either viral vectors and/or genetically modified cultured cells became inevitable. Despite the early exposition of the concept of gene therapy, progress awaited the advent of recombinant DNA technology. The lack of trustworthy techniques did not stop many researchers from attempting to transfer genes into cells in culture, animals, and humans. Viral genomes were used for the development of the first relatively efficient methods for gene transfer into mammalian cells in culture. In the late 1970s, early transfection techniques were combined with selection systems for cultured cells and recombinant DNA technology. With the development of retroviral vectors in the early 1980s, the possibility of efficient gene transfer into mammalian cells for the purpose of gene therapy became widely accepted.

High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA.

We have previously shown that the intramuscular injection of naked plasmid DNA enables foreign gene expression in muscle. Further studies showed that the intravascular delivery of naked plasmid DNA enables high levels of expression not only in muscle but also in hepatocytes. For the liver, this technique required injection directly into the liver vessels (portal vein, hepatic vein, or bile duct) and occlusion of outflow. The present study now demonstrates that high levels of plasmid DNA expression in hepatocytes can be easily obtained by tail vein injections. The highest levels of expression are achieved by rapidly injecting the plasmid DNA in large volumes, approximately 2.5 ml. This technique has great potential for a wide variety of laboratory studies.

Gene transfer into mammalian cells using histone-condensed plasmid DNA.

A recombinant histone (NLS-H1) containing both the SV40 large T antigen nuclear localization signal and the carboxy-terminal domain of human histone H1(0) was produced in bacteria. NLS-H1-plasmid DNA complexes, in the presence of chloroquine, mediated reporter gene transfer into cultured cells with similar efficiencies as plasmid DNA-cationic lipid (lipofectin) complexes. NIH-3T3 or COS-7 cells transfected with NLS-H1-plasmid DNA-lipofectin complexes expressed at least 20 times more luciferase or had at least 2.5 times more beta-galactosidase-positive cells than those transfected with plasmid DNA-lipofectin complexes. Foreign gene expression was also improved by other DNA-binding proteins and cationic lipid formulations, yet the greatest enhancement was obtained with complexes containing either NLS-H1 or calf thymus histone H1. Histone H1-plasmid DNA-lipofectin complexes were internalized by a greater number of cells than plasmid DNA-lipofectin complexes.

Efficient expression of naked dna delivered intraarterially to limb muscles of nonhuman primates.

We have previously shown that the intraarterial delivery of naked plasmid DNA (pDNA) into the femoral artery of rats leads to high levels of foreign gene expression throughout the muscles of the hindlimb. The present study shows that the procedure can also enable high levels of foreign gene expression throughout the limb skeletal muscles in rhesus monkeys. The average luciferase expression in the target muscle was 991.5 +/- 187 ng/g for the arm and 1692 +/- 768 ng/g for the leg; compared with 780 ng/g in rat hindlimb. Large numbers of beta-galactosidase-positive myofibers were found in both leg and arm muscles, ranging from less than 1% to more than 30% in various muscles, with an average of 6.9%. The nonhuman primates tolerated the procedure without significant adverse effects in skeletal muscles, arteries, or other organs. Other studies in immunosuppressed rats indicated that stable expression is possible. These results suggest that the procedure is likely to enable efficient and stable gene expression in human muscle without substantial toxicity.

A plasmid-based self-amplifying Sindbis virus vector.

Sindbis virus was used as a self-amplifying eukaryotic expression vector. A recombinant cDNA genome of this (+)-strand RNA virus was placed under the transcriptional control of a Rous sarcoma virus LTR (RSV) promoter. Transfection of this plasmid construct into mammalian cell lines (3T3, HepG2, and 293 cells) resulted in expression of the luciferase reporter gene. High-expression levels were also measured after transfection into primary rat myoblasts. In differentiated myotubes, expression levels generated by the Sindbis virus vector were up to 200 times higher than those obtained with a conventional RSV expression vector. In vivo expression was detected after injection of plasmid DNA into mouse quadriceps. In vivo expression was transient and undetectable by day 16. This self-amplifying expression vector can be used for generating high-level expression of transgenes in vitro and in vivo. Its transient nature in vivo could allow for safe, short-term delivery of gene products in gene therapy protocols. It should facilitate the study of Sindbis and other RNA viruses.

Expression of naked plasmid DNA injected into the afferent and efferent vessels of rodent and dog livers.

A variety of reporter genes within plasmid constructs were injected into the afferent and efferent vessels of the liver in mice, rats, and dogs. Efficient plasmid expression was obtained following delivery via the portal vein, the hepatic vein, and the bile duct. The use of hyperosmotic injection solutions and occlusion of the blood outflow from the liver substantially increased the expression levels. Combining these surgical approaches with improved plasmid vectors enabled uncommonly high levels of foreign gene expression in which over 15 microg of luciferase protein/liver was produced in mice and over 50 microg in rats. Equally high levels of beta-galactosidase (beta-Gal) expression were obtained, in that over 5% of the hepatocytes had intense blue staining. Expression of luciferase or beta-Gal was evenly distributed in hepatocytes throughout the entire liver when either of the three routes were injected. Peri-acinar hepatocytes were preferentially transfected when the portal vein was injected in rats. These levels of foreign gene expression are among the highest levels obtained with nonviral vectors. Repetitive plasmid administration through the bile duct led to successive events of foreign gene expression. The integration of these findings into laboratory and clinical protocols is discussed.

Direct gene transfer into nonhuman primate myofibers in vivo.

Previously, we showed that rodent muscle has the ability to take up and express plasmid genes injected intramuscularly. This study now demonstrates that nonhuman primate muscle also has this ability to express injected plasmids. A scaled-up version of the standard large preparation of plasmid DNA allowed several tens of milligrams of CCC plasmid DNA to be relatively easily produced and administered to monkeys. After the injection of the E. coli beta-galactosidase reporter gene in pRSVLac-Z, foreign gene expression was localized to both type I and type II myofibers. The luciferase reporter gene in pRSVL was used to quantify the amount of expression. The multiple implantation of plasmid DNA pellets was more efficient in expressing luciferase than the injection of DNA in normal saline. Luciferase activity persisted for at least 4 months after injection. However, the luciferase expression was considerably less than that in rodents. Preliminary studies explored why expression was less in monkeys. Of particular interest was the increased thickness of the perimysium of monkeys as compared to that in rodents. This increased connective tissue may decrease delivery of the plasmid DNA to the myofibers. Anti-nuclear or anti-DNA antibodies were not observed, even after repetitive DNA administrations, and no adverse effects were observed in any of the monkeys.

Self-amplifying expression from the T7 promoter in 3T3 mouse fibroblasts.

To increase the levels of exogenous or foreign gene expression in mammalian cells, this study sought to develop an 'autogene' that will self-amplify. An autogene plasmid, pT7-G1, containing the T7 phage RNA polymerase-encoding modified gene (G1) under control of its cognate T7 promoter, was only obtained when the plasmid contained the encephalomyocarditis (EMC) untranslated sequence. In vitro transcription and translation studies confirmed that both the T7 promoter and the G1 gene were completely functional in the pT7-G1 plasmid. Expression from pT7-G1 was initiated in vivo either by co-transfection with its in vitro transcript or by transfection into NIH3T3 cell lines that stably expressed T7 RNA polymerase enzyme. Use of the pT7-G1 autogene enabled an approx. 50-fold increase in foreign protein production. Northern analysis suggested that this increased expression resulted from the self-amplification of the autogene. By allowing greater expression in cell lines with low T7 RNA polymerase expression, the pT7-G1 plasmid increases the usefulness of the T7 gene system for expression within mammalian cells.

High-efficiency protein synthesis from T7 RNA polymerase transcripts in 3T3 fibroblasts.

After NIH3T3 cells constitutively expressing T7 RNA polymerase were transfected (+ Ca.phosphate) with a circular DNA containing the firefly luciferase(Luc)-encoding gene (luc) 3' to the encephalomyocarditis (EMC) virus 5'-untranslated sequence and T7 promoter, Luc protein comprising approx. 20% of total cellular protein was obtained. After similar transfection of an analogous construct containing the lacZ gene into the same cell line, at least 50% of the cells produced beta-galactosidase. Fibroblasts lipofected with uncapped RNA transcripts containing EMC sequence expressed the reporter genes as efficiently as capped transcripts. A novel approach was used to generate RNA transcripts containing poly(A) at its very 3' end. RNA from a luc vector with a poly(A) sequence at the very 3' end produced 20-fold more Luc than the RNA from the same vector with an additional 3' nonpoly(A) sequence. These results suggest that this T7 RNA polymerase expression system will be useful for the efficient production of proteins in mammalian cells.

Wilm's Tumor: An Update.

Major advances have been made against Wilms' tumor as a result of treatment methods developed by single institutions that then have been confirmed and extended by national cooperating groups. Better survival rates have been achieved, and therapy has been refined so that treatment can be reduced in early stage disease without jeopardizing tumor control. This results in fewer short- and long-term complications, an especially important consideration in children. Their organs and tissues are vulnerable to anti-mitotic treatments such as chemo- and radiotherapy, that can produce disabling if not lethal dysfunctions. This progress has been the result of the cooperative efforts by multiple specialists, and provides evidence of the value of such integrated studies. They have changed the outlook from a 90% death rate in the early years of this century to the 90% survival rate now possible with modern management.

Naked DNA transport and expression in mammalian cells.

The tremendous promise of gene therapy is stymied by inefficient gene transfer. Non-viral methods of transferring genes directly into tissues in situ have great potential for solving this problem. One attractive approach for non-viral direct gene transfer is the use of naked DNA. Previously, it was thought that naked DNA was able to express in only muscle and at low efficiency. We have recently found that naked DNA delivered intraportally can express at high levels in hepatocytes. Other non-viral methods of gene transfer are under development and these include ternary complexes of histone protein and liposomes. Besides trial-and-error approaches, the mechanism of the nuclear entry of plasmid DNA, a critical step, has been under intense study. It is anticipated that further advances in the efficiency of direct, non-viral gene transfer will enable its use in clinical settings in the near future.