Autosomal recessive osteopetrosis (ARO) is a paradigm for genetic diseases that cause severe, often irreversible, defects before birth. In ARO, osteoclasts cannot remove mineralized cartilage, bone marrow is severely reduced, and bone cannot be remodeled for growth. More than 50% of the patients show defects in the osteoclastic vacuolar-proton-pump subunit, ATP6a3. We treated ATP6a3-deficient mice by in utero heterologous hematopoietic stem cell (HSC) transplant from outbred GFP transgenic mice. Dramatic phenotype rescue by GFP osteoclasts was obtained with engraftment, which was observed in most cases. Engraftment survived for variable periods. Recipients were not immunosuppressed, and graft-versus-host disease was not observed in all pups born after in utero treatment. Thus, differentiation of unmatched HSC transplanted in utero is sufficient to prevent fatal defects in ARO and may prevent complications of ARO unresponsive to conventional bone marrow transplantation. The presence of defective cells is not a barrier to the rescue of the phenotype by donor HSC.autosomal recessive osteopetrosis ͉ bone marrow transplant ͉ prenatal therapy

Tumor growth and metastasis are angiogenesis-dependent. The possibility of inhibiting tumor growth by interfering with the formation of new vessels has recently raised considerable interest. We previously reported that it is possible to inhibit primary tumor growth and metastasis in a transgenic model of spontaneous breast tumor, which shows many similarities to its human counterpart (including ability to metastasize) by intratumoral administration of a DNA conKeywords: gene therapy; breast adenocarcinoma; metastasis; transgenic mice; anti-angiogenesis; liposomesIt is now widely recognized that the development of new blood vessels (angiogenesis) is necessary to sustain tumor growth, invasion and metastasis. At some point during tumor growth, cancer cells acquire the ability to activate the quiescent vasculature to produce new blood vessels via a so-called 'angiogenic switch '. 1,2 Although the need for recruiting new blood vessels to the tumor has been know for more than two decades, the possibility of treating malignancies by acting on their vasculature has become popular only since the isolation of endogenous angiogenesis inhibitors, which dramatically affect cancer growth in experimental murine systems. Highly promising results on experimental tumors in rodents using these inhibitors as purified proteins as well as transduced genes, alone or in combination with traditional therapies have been reported. [3][4][5][6][7] The establishment of a dormancy status, which in some cases persists even after the suspension of therapy, has been obtained with these gene products, 5 and this, together with the absence of drug resistance, 8 raised the possibility that the anti-angiogenic effects could be useful in the treatment of human tumors.The achievement of the inhibition of local tumor growth is the usual end-point of these therapeutical attempts, but in humans it is the metastatic spread that is responsible for the majority of cancer-related deaths. Therefore, to be really useful in the human context, any new approach must be evaluated in its ability to interfere Correspondence: MG Sacco, ITBA CNR, Via F lli Cervi, 93, 20090 Segrate (MI), Italy Received 17 August 2000; accepted 11 October 2000 struct carrying the murine angiostatin cDNA driven by liposomes. Here we report that it is also possible to achieve this goal by a systemic (intraperitoneal) delivery of therapeutic DNA constructs carrying genes coding for mouse and human anti-angiogenic factors which include angiostatin, endostatin and TIMP-2. These findings may be relevant to the design of therapeutic interventions in humans. Gene Therapy (2001) 8, 67-70. with tumor cell invasion and metastasis, a therapeutical target which is rarely, if ever, investigated at the experimental level. Unfortunately, most of the results in experimental oncology have been obtained on transplantable tumors which are usually generated by inoculating highly malignant cultured cells, which rapidly grow in the site of injection and become the target for therapeutic intervention. ...

Mutations in the CD40 ligand (CD40L) are responsible for human hyper immunoglobulin M (IgM) syndrome. The absence of the interaction between CD40L, expressed by T lymphocytes, and the CD40 receptor present on the surface of B cells is responsible for the inability of B cells to carry out the isotype switch from IgM to the other Ig classes. This leads to a fatal immunodeficiency for which no cure exists. For these reasons, the CD40L gene is a good candidate for gene therapy studies. To investigate the possible effects of the expression of this tightly regulated gene in vivo, we produced transgenic mice in which CD40L expression was deregulated. Widespread ectopic expression appears to be lethal. Overexpression in mature T cells is compatible with life, but in one-third of the cases, mice developed atypical lymphoid proliferations which, occasionally, progressed into frank lymphomas. Even though gene therapy is one of the most promising approaches to cure human hyper IgM syndrome, these results suggest that when we modify very tightly regulated genes such as cytokines or other growth factors, particular care has to be taken to avoid excessive stimulation of the target cells.

Transgenic mice for genotoxicity testing have been developed, although no such models have been produced for the evaluation of toxic, nongenotoxic chemical compounds. We have developed a transgenic mouse model for the analysis of toxic inorganic compounds. We engineered a mouse lineage with the human growth hormone (hGH) gene under the control of the human hsp70 promoter, in which a plasma-detectable hGH response can be elicited by exposure to heat shock. In primary cell cultures from these mice, hGH release was observed following treatment with several toxic inorganics. Transgenic mice injected intraperitoneally with sodium arsenite, cadmium chloride, copper sulphate, or methylmercurium chloride showed significant hGH levels in plasma.

Breast cancer in women is a major health problem, causing a great deal of suffering and a high number of deaths. So far, early diagnosis followed by surgery is the only way to cure the disease, while therapeutic protocols for women at an advanced stage of the disease are still unsatisfactory and, in the long term, metastatic breast cancer is still associated with high treatment failure. 1 In humans, both chemotherapy and hormonal approaches for the management of the advanced disease are of great clinical relevance. Various drugs, including taxol, are active against disseminated breast cancer. 2 In addition, in patients with estrogen receptor positive tumors, antiestrogens, and tamoxifen in particular, have been widely used. 3 Many randomized trials have shown that postsurgical tamoxifen (TAM) treatment significantly reduces the growth of hormone receptor-positive breast cancer. Recently, the efficacy of TAM for chemoprevention of breast tumors has been evaluated, and the data from the Breast Cancer Prevention Trial have demonstrated that TAM administration reduces the incidence of estrogen receptor-positive, but not receptor-negative tumors. 4,5 More recently, inhibition of tumor angiogenesis was investigated as a therapeutic strategy that can inhibit Correspondence: MG Sacco, CNR-ITB, Via Fratelli Cervi, 93, 20090 Segrate (MI)

Treatments available to women with locally advanced breast cancer are unsatisfactory, since most patients succumb to metastatic spread. Therefore, there is a need to devise novel therapeutic combinations that effectively inhibit metastatization and to test them in animal models of breast cancer showing strong similarities with their human counterpart, including the ability to give rise to metastases. With these considerations in mind, tamoxifen (TAM), 4-hydrotamoxifen (4-HT) or liposome-complexed DNA constructs coding for antiangiogenic/anti-invasion proteins (angiostatin, TIMP-2, IFN-a 1 , sFLT-1) were individually administered to MMTVneu transgenic mice. Significant inhibition of primary tumor growth was obtained with TAM (40% inhibition, P¼0.049), angiostatin (85% inhibition, P¼0.001) and TIMP-2 (60% inhibition, P¼0.015). No lung metastasis was observed in any of these treated mice at 5 months, compared with a rate of 70% in control groups. These observations were the basis for designing a combined treatment with all these compounds. The association of angiostatin, TIMP-2 and TAM was greatly effective at the primary tumor level (90% inhibition, P¼0.01). Moreover, all the mice treated with this association were metastasis free at a time point (6 months) in which seven out of nine control mice were either dead from disseminated cancer or showed lung metastasis. This combined therapy could become an important component of anticancer therapy in humans.

Gene therapy approaches to the treatment of experimental mice. The size of IL-4 inoculated tumors on the right side cancer are usually based on established neoplastic cell was significantly smaller than that of controlateral lines which are manipulated in vitro and subsequently untreated tumors, suggesting a local effect of IL-4. In transplanted in host animals. However, the relevance of addition, the non-injected tumors on the left side of treated these artificial models to the biology and therapy of human animals were significantly smaller than those arising in tumors is uncertain. We have previously validated an control transgenic mice, suggesting that IL-4 can also experimental model based on MMTV-neu transgenic mice inhibit tumor growth systemically. These findings suggest in which breast tumors arise spontaneously in 100% of anithat IL-4 gene transfer can significantly reduce the growth mals and have many features in common with their human rate of spontaneously arising breast tumors and that counterpart, including the involvement of the neu oncoimmune-based gene therapy could efficiently complement gene and the ability to metastatize. In this article we report other approaches based on different mechanisms, such as the effect of intratumoral, retrovirus-mediated, IL-4 suicide gene transfer or antisense technology. expression on the growth of breast tumors arising in these

Mutations in the CD40 ligand (CD40L) are responsible for human hyper immunoglobulin M (IgM) syndrome. The absence of the interaction between CD40L, expressed by T lymphocytes, and the CD40 receptor present on the surface of B cells is responsible for the inability of B cells to carry out the isotype switch from IgM to the other Ig classes. This leads to a fatal immunodeficiency for which no cure exists. For these reasons, the CD40L gene is a good candidate for gene therapy studies. To investigate the possible effects of the expression of this tightly regulated gene in vivo, we produced transgenic mice in which CD40L expression was deregulated. Widespread ectopic expression appears to be lethal. Overexpression in mature T cells is compatible with life, but in one-third of the cases, mice developed atypical lymphoid proliferations which, occasionally, progressed into frank lymphomas. Even though gene therapy is one of the most promising approaches to cure human hyper IgM syndrome, these results suggest that when we modify very tightly regulated genes such as cytokines or other growth factors, particular care has to be taken to avoid excessive stimulation of the target cells.