Analysis of testes and semen from rabbits treated by intravenous injection with a retroviral vector encoding the human factor VIII gene: no evidence of germ line transduction.

In a phase 1 clinical trial, we are evaluating a murine leukemia virus (MuLV)-based retroviral vector encoding the human factor VIII gene [hFVIII(V)], administered intravenously, as a therapy for hemophilia A. Preclinical biolocalization studies in adult rabbits revealed vector-specific PCR signals in testis tissue at low levels. In follow-up animal studies we used PCR to (1) estimate the frequency with which a given cell in testis tissue is transduced, and (2) determine whether a positive PCR signal could be detected in semen samples from animals treated with hFVIII(V). Using the 99% confidence bound, results indicate that the probability that a given cell within the testis was transduced is less than 1/709,000 (97 days after treatment). This probability decreased with time after hFVIII(V) administration. Moreover, the rate of provector sequence detection in semen samples collected weekly throughout two cycles of spermatogenesis was 3/4281 reactions (0.07%), which is lower than the rate of false positives (1/800, 0.125%) observed for control animals. Using PCR assays with single-copy sensitivity, we have shown that the small number of transduced cells present in testis tissue does not give rise to detectable transduced cells in semen.

Safety evaluation of gene therapy viral vectors.

Gene therapy has engendered interest in scientists, regulators and the public alike, because it represents the application of current expertise in the areas of cellular and molecular biology to the specific modification of cell function for the purposes of ameliorating disease. As scientific advances broaden the application of gene therapy, it is increasingly important to adequately evaluate the safety of these agents prior to clinical trials. A number of guidance documents have been published which address strategies for preclinical evaluation of gene therapy candidates. Some aspects of preclinical safety evaluation are similar to the development of more traditional drugs, while others are unique, entwined with the molecular and cellular biology needed to accurately describe a gene therapy's activity and distribution. This review provides a summary of some of the practical issues to consider in the preclinical evaluation of gene therapy viral vectors. These range from the demonstrated or theoretical toxicities of a specific vector class, to the risk of inadvertent germline transfer and insertional mutagenesis.

Activation of human monocytes with lipopolysaccharide induces metallothionein expression and is diminished by zinc.

The metal-binding protein metallothionein (MT) confers resistance to the toxic effects of metals. Although a role for MT in metal homeostasis and protection against toxic free radicals has been suggested, no clear physiological function has been established. The ability of human monocytes to be activated by bacterial lipopolysaccharide (LPS) treatment provided a model to investigate the effect of zinc on both cellular activation (H2O2 production) and MT expression. In both primary human monocytes and a monocyte-derived cell line (THP-1), LPS induced activation and MT expression; it did not induce MT expression in nonmonocyte human cells. Treatment of THP-1 cells with nontoxic zinc levels increased MT accumulation. Subsequent treatment with LPS resulted in a decrease in both MT mRNA and protein levels and inhibited the ability of THP-1 cells to undergo the respiratory burst. Pretreatment with cadmium had the same inhibitory effect. We conclude that MT expression is associated with monocyte activation, and exposure to zinc or cadmium interferes with the ability of monocytes to respond to activation signals. Metallothionein may play a role in that response.

Differential toxicity of cisplatin, carboplatin, and CI-973 correlates with cellular platinum levels in rat renal cortical slices.

The differential toxicity of the platinum-containing compounds, cisplatin, carboplatin, CI-973, and transplatin, was investigated in renal cortical slices over a 24-hr period. Platinum accumulation was measured to assess whether platinum levels correlated with inhibition of specific cell functions (accumulation of organic ions, protein synthesis) or potassium loss. The earliest indicator of toxicity was decreased accumulation of the organic anion, para-aminohippuric acid (PAH), which preceded a decrease in the accumulation of the organic cation, tetraethyl-ammonium (TEA), and protein synthesis. Cisplatin (1 mM) and CI-973 (3 mM) treatment reduced PAH accumulation within 2 hr; carboplatin (3 mM) decreased PAH at 6 hr. TEA accumulation was absent in slices incubated for 6 hr or longer in 1 mM cisplatin, or for 24 hr in 3 mM carboplatin or CI-973. Protein synthesis was inhibited 80, 75, and 87% in slices treated for 24 hr with 100 microM cisplatin, 1 mM carboplatin, and 1 mM CI-973, respectively, compared to control. Intracellular potassium levels, which decreased between 6 and 24 hr, were the least sensitive indicator of cell damage. Transplatin, which lacks antitumor activity and is nonnephrotoxic in vivo, effectively decreased protein synthesis, intracellular potassium, and organic ion accumulation in rat renal cortical slices in vitro. The concentration of slice-associated platinum following treatment with cisplatin, carboplatin, CI-973, and transplatin increased with time and concentration. Inhibition of protein synthesis and loss of intracellular potassium correlated with increased total cellular platinum, indicating that platinum compounds negatively affect cell function and viability. The relative toxicity of these compounds in rat renal cortical slices was cisplatin = transplatin > CI-973 > carboplatin.

Antisense down-regulation of metallothionein in a human monocytic cell line alters adherence, invasion, and the respiratory burst.

A human monocyte-derived cell line (THP-1) was used as a model to investigate the role of metallothionein (MT) in the cellular physiology of resting and activated monocytes. MT protein levels were reduced in THP-1 cells by transient transfections with an antisense MT expression vector. Antisense mouse MT-1 RNA was constitutively expressed under the control of the H-2Kb (mouse major histocompatibility complex I) promoter and could be further induced by lipopolysaccharide (LPS) treatment. THP-1 cells expressing antisense MT RNA (aMT-THP-1) had a 30% reduction in MT protein levels. In the absence of LPS treatment, aMT-THP-1 cells demonstrated increased production of H2O2 concurrent with enhanced adherence and invasiveness compared to cells transfected with the control vector (cv-THP-1). Treatment of aMT-THP-1 cells with LPS depressed these activation-associated responses and further reduced the level of MT protein. cv-THP-1 cells activated by LPS produced high levels of H2O2 and adhered to and invaded a reconstituted basement membrane. In addition to increasing cadmium sensitivity, diminished MT levels affected broad-ranging processes associated with resting and activated monocyte function. Thus, metallothionein plays an important physiological role in cells in addition to its role in detoxification of heavy metals.

Critical subcellular targets of cisplatin and related platinum analogs in rat renal proximal tubule cells.

The clinical usefulness of chemotherapeutic agents containing the platinum moiety is often limited by their nephrotoxicity. To investigate the mechanism of nephrotoxicity, and to assess the effects of platinum analogs on specific organelles and basal protein synthesis, biochemical and ultrastructural analyses were performed in rat renal proximal tubule cells (RPTCs). Neutral red (NR) uptake was used to measure lysosomal function, and conversion of MTT to formazan used to assess mitochondrial function. Despite their differential toxicity, cisplatin, carboplatin and CI-973 caused similar progressive inhibition of specific functions, suggesting they may share a common mechanism of nephrotoxicity. Protein synthesis was the earliest indicator of toxicity, followed by NR uptake and MTT conversion. Fluorescent probes for lysosomes (acridine orange) and mitochondria (rhodamine 123) confirmed that cisplatin's toxicity to RPTCs was delayed and cumulative. Condensation of nucleolar components and fragmentation of RER were observed in RPTCs treated for as little as two hours. Since the nucleolus is the site of ribosome biogenesis, the early inhibition of protein synthesis by cisplatin may arise from disruption of this region. In contrast, mitochondrial dysfunction and swelling were late-stage events, and are therefore unlikely to be the primary targets of nephrotoxic platinum compounds.