Jon Wolff

Grafting genetically modified cells to the brain: possibilities for the future.

Diagnostic and therapeutic approaches to disorders of the central nervous system (CNS) are particularly difficult to develop because of the relative inaccessibility of the mammalian brain to study and chemical treatment, the complexity and interconnectedness of CNS subsystems, and the profound and continued lack of fundamental understanding of the relationship between structure and function in the CNS. Neural grafting in the CNS has recently suggested a potential approach to CNS therapy through the selective replacement of cells lost as a result of disease or damage. Independently, studies aimed at direct genetic therapy in model systems have recently begun to suggest conceptually new approaches to the treatment of several kinds of human genetic disease, especially those caused by single-gene enzyme deficiencies. We suggest that a combination of these two approaches, namely the grafting into the CNS of genetically modified cells, may provide a new approach toward the restoration of some functions in the damaged or diseased CNS. We present evidence for the feasibility of this approach, including a description of some current techniques for mammalian cell gene transfer and CNS grafting, and several possible approaches to clinical applications.

Effects of Ascorbic Acid in Alkaptonuria: Alterations in Benzoquinone Acetic Acid and an Ontogenic Effect in Infancy

ABSTRACT: The effects of ascorbic acid on the excretion of homogentisic acid and its derivative benzoquinone acetic acid were studied in two adults and three infants. The administration of relatively large amounts of ascorbic acid to the adults was followed by a disappearance of benzoquinone acetic acid from the urine, whereas the level of excretion of homogentisic acid did not change. This could have relevance to the pathogenesis of ochronotic arthritis. In the 4-mo-old infant and the 5-mo-old infant ascorbic acid in the urine may have doubled the amount of homogentisic acid, presumably through an effect on the immature p-hydroxyphenylpyruvic acid oxidase. Dietary reduction of the intake of tyrosine and phenylalanine substantially reduced the excretion of homogentisic acid.

Deficiency of Carnitine Palmitoyltransferase I

Defective activity of carnitine palmitoyltransferase I was demonstrated in fibroblasts derived from a patient with hypoketotic hypoglycemia. The level of activity observed was approximately 10% of the control mean. Oxidation of palmitate by intact fibroblasts was reduced to 5% of control values. The patient presented at age 14 months with seizures and was found to have marked hypoglycemia and no ketones in the urine. In response to fasting, she developed hypoglycemia, but the curves for acetoacetate and 3-hydroxybutyrate were flat. Administration of medium-chain triglycerides relieved the hypoglycemia and generated a brisk ketogenesis. (J Child Neurol 1989;4:197-202).

Proximal renal tubular acidosis in methylmalonic acidemia.

A patient with methylmalonic acidemia was found to have a persistent hyperchloremic acidosis. Investigation documented the presence of a proximal renal tubular acidosis. Between 14 and 18 months of age the urinary pH was as high as 8.0 when the serum bicarbonate was 17 mEq/liter and the threshold for bicarbonate was at 16-17 mEq/liter. When restudied at 33 months of age, the threshold had risen to 20 mEq/liter, but this was still abnormal and supplemental treatment was required to keep the serum concentration of bicarbonate above 20 mEq/liter. It is postulated that organic acid metabolites which accumulate in this and related disorders may interfere with renal tubular function as has been shown for maleic acid in experimental animals.

Myopathy in an infant with a fatal peroxisomal disorder

An infant with neonatal adrenoleukodystrophy experienced extreme hypotonia and virtually continuous convulsions at four months of age and died. Light and electron microscopic examination revealed evidence of myopathy and the presence of mitochondrial inclusions. Concentrations of very long-chain fatty acids were elevated in blood and fibroblasts and the oxidation of 14C-labeled fatty acids was defective. Urinary pipecolic acid content was increased. Activity of the peroxisomal dihydroxyacetone phosphate acyltransferase, which catalyzes the first step in plasmalogen synthesis, was decreased.

Pharmacologic amino acid acylation in the acute hyperammonemia of propionic acidemia

An infant with propionic acidemia presented at one month of age with hyperammonemic coma. Treatment by two double-volume exchange transfusions did not have an appreciable effect, but hemodialysis led to a substantial reduction of the serum concentration of ammonia on two occasions. Nevertheless, continued therapy with sodium benzoate, arginine-HCl, carnitine and lactulose did not have any observable effect on the blood concentration of ammonia. Treatment with sodium phenylacetate was followed by a reduction in serum concentrations of ammonia to normal levels which were maintained. These observations demonstrate the dramatic reduction in serum concentrations of ammonia that may be obtained in patients with organic acidemia by hemodialysis. They suggest that pharmacologic acylation therapy with phenylacetate may be of lasting benefit in the management of this complication.

Chapter 86 Implantation of genetically engineered cells to the brain

Publisher Summary This chapter discusses the combination of gene transfer via efficient viral vectors, followed by intracerebral grafting of the genetically modified cells may constitute an effective approach to some disorders of central nervous system (CNS) function. Several prerequisites for a human disease to be suitable as a candidate recipient for transplantation of genetically engineered cells are also discussed, including the pathogenesis and pathophysiology of the disease must be sufficiently well understood for the identification of the relevant gene product to be introduced into defective cells, the relevant gene must be well characterized and available as a clone, or ideally, an animal model should be available. However, the major difficulty with most human diseases of the CNS is that these criteria cannot be satisfied, because the exact mechanisms of pathophysiology and pathogenesis in most human genetic diseases, such as phenylketoneuria and Lesch-Nyhan disease, are not adequately understood. Although, the present models are all very complex, and truly effective therapy is likely to be extremely difficult to achieve, but it seems probable that a number of conceptually new approaches to these previously inaccessible diseases will become feasible, and that such new approaches will include the implantation of genetically modified cells.

Epitope insertion into the human hypoxanthine phosphoribosyltransferase protein and detection of the mutant protein by an anti-peptide antibody

The translational stop codon TAA of the human hypoxanthine phosphoribosyltransferase (HPRT) cDNA has been changed to GAA by site-specific mutagenesis. This modification extends the open reading frame to a downstream stop codon and results in the addition of a unique negatively charged hexapeptide to the C terminus of human HPRT protein. The mutated cDNA was transferred into HPRT-deficient rodent cells by retroviral vector infection, and the expressed enzyme was found to be fully active. An antibody against a synthetic octapeptide corresponding to the mutated HPRT C terminus precipitated the HPRT protein specifically from cells infected with the mutant virus and not infected with the wild-type HPRT virus. The technique of inserting a novel epitope into a protein by site-directed mutagenesis should be generally applicable in studies of the regulation of gene expression in vitro and in vivo.

Alanine Decreases the Protein Requirements of Infants with Inborn Errors of Amino Acid Metabolism

Supplementation with alanine was found to increase growth in weight and nitrogen balance in 5 infants with a variety of inborn errors of amino acid metabolism receiving diets restricted in protein. The addition of alanine to the regimen led to a mean increase in weight of 15 g/day. This and the increased nitrogen balance of 15 mg/kg/day were highly significant statistically. In addition a dose-response effect of alanine was observed. The effect of alanine was compared with that of a supplemental mixture of essential and non-essential amino acids, lacking only those considered to be toxic in these patients. Alanine at 0.05 g/kg was as effective in promoting growth in weight as 1.05 g/kg of the amino acid mixture, while 0.25 g/kg of alanine was more effective than 0.70 g/kg of the amino acid mixture. The protein sparing anabolic effect of alanine is thought to be a reflection of the alanine glucose cycle.

Adult Mammalian Hepatocyte as Target Cell for Retroviral Gene Transfer: A Model for Gene Therapy

Gene therapy models have recently been developed based on the repopulation of a major organ, such as mammalian bone marrow, with cells genetically modified in vitro by infection with retroviral vectors (1-4). Another important target organ for therapeutically useful gene transfer is the liver. Unfortunately, the murine leukemia viruses (MLV) used to construct most retroviral vectors do not infect newborn, adult, or regenerating liver cells in vivo (5-8), although fetal liver can be infected and continues to express retrovitally transduced genes postnatally (7, 8). We now show that adult primary rat hepatocyte cultures (9, 10) are in fact susceptible to infection with MLV-based retroviral vectors during a period of transient dedifferentiation in vitro. Primary hepatocyte cultures exhibit many adult liver-specific functions--including gluconeogenesis and the expression of albumin, alcohol dehydrogenase, and type I pyruvate kinase--during their early lag (days 0-2) and late stationary (after day 11) phases of culture (10 16). During the intervening phase of logarithmic growth, these mature liver-specific functions are transiently but markedly decreased, and fetal hepatocyte functions such as alphal-fetoprotein and type III pyruvate kinase are expressed (12). Hepatocyte cultures were exposed at varying times during their growth cycle to a retroviral vector expressing a mutagenized but fully functional human hypoxanthine guanine phosphoribosyl transferase (HPRT) (Fig. 1, LSAPALM) (J.-K. Yee et al., in preparation). Human HPRT enzymic activity was detected in isoelectric focusing polyacrylamide gels (17) under conditions that distinguish it from the endogenous rat enzyme by its altered isoelectric point. Twoto three-dayold cultures infected with 5 x 1 0 6 infectious units of the HPRT vector exhibited human HPRT enzymic activity on day 14 postplating, as indicated by the arrows in Fig. 2A (lane a). The transduced human HPRT activity was at least 10-25% of the endogenous rat HPRT enzymic activity, equivalent to the high levels achieved in primary rat fibroblasts (Fig. 2, lane c) and human lymphoblasts (18). Since the upper bands in Fig. 2A (lanes a and c) may also contain human HPRT multimers (17), the actual transduced levels of human HPRT enzymic activity may be even higher. Figure 2B shows that human HPRT activity was present only when the hepatocyte cultures were infected during the early proliferative, dedifferentiated period of growth (days 2-3). Very low levels of human HPRT expression, not evident in Fig. 2B, were also detected on days 1 and 5, but not at any other times of infection. In addition, quiescent hepatocytes on day 1 1 can also be stimulated to reinitiate DNA synthesis and undergo additional cell division by exposure to fresh medium containing rat serum or polypeptide mitogens, mimicking the regenerating adult liver following partial hepatectomy (9, 19).