Viral and non-viral gene therapy partially prevents experimental cisplatin-induced neuropathy.

Sensory neuropathies are a frequent and dose-limiting complication resulting from treatment with cisplatin. Neurotrophin-3 (NT-3) promotes the survival of the large fiber sensory neurones that are impaired in cisplatin-induced neuropathy, and may therefore serve as a preventive agent. However, the short half-life of recombinant NT-3 after systemic administration limits its clinical applications. We compared two muscle-based gene transfer strategies for the continuous delivery of NT-3 to the bloodstream in an experimental model of cisplatin-induced neuropathy. Electrophysiological studies showed that the intramuscular injection of an adenovirus encoding NT-3 partially prevented the cisplatin-induced increase in sensory distal latencies. Similar effects were observed in cisplatin-treated mice that received intramuscular injections of a plasmid encoding NT-3 associated with in vivo electroporation. The two techniques were well tolerated and induced only slight muscle toxicity. Measurement of renal function, weight and survival showed that neither technique increased the toxicity of cisplatin. Our study shows that gene therapy, using either a viral or a non-viral vector, is a promising strategy for the prevention of cisplatin-induced neuropathy.

Partial prevention of cisplatin-induced neuropathy by electroporation-mediated nonviral gene transfer.

Cisplatin-induced sensory peripheral neuropathy is the dose-limiting factor for cisplatin chemotherapy. We describe the preventive effect of NT-3 delivery, using direct gene transfer into muscle by in vivo electroporation in a mouse model of cisplatin-induced neuropathy. Cisplatin-induced neuropathy was produced by weekly injections of cisplatin (five injections). Two doses of plasmid DNA encoding murine NT-3 (pCMVNT-3) were tested (5 and 50 microg/animal/injection). Cisplatin-treated mice were given two intramuscular injections. The first injection of pCMVNT-3 was given 2 days before the first injection of cisplatin and the second injection 2 weeks later. Six weeks after the start of the experiment, measurement of NT-3 levels (ELISA) demonstrated significant levels both in muscle and plasma. We observed a smaller cisplatin-related increase in the latency of the sensory nerve action potential of the caudal nerve in pCMVNT-3-treated mice than in controls (p < 0.0001). Mean sensory distal latencies were not different between the 5- and 50- microg/animal/injection groups. Treatment with gene therapy induced only a slight muscle toxicity and no general side effects. Therefore, neurotrophic factor delivery by direct gene transfer into muscle by electroporation is of potential benefit in the prevention of cisplatin-induced neuropathy and of peripheral neuropathies in general.

Continuous delivery of neurotrophin 3 by gene therapy has a neuroprotective effect in experimental models of diabetic and acrylamide neuropathies.

Neurotrophic factors (NFs) are promising agents for the treatment of peripheral neuropathies such as diabetic neuropathy. However, the value of treatment with recombinant NF is limited by the short half-lives of these molecules, which reduces efficiency, and by their potential toxicity. We explored the use of intramuscular injection of a recombinant adenovirus encoding NT-3 (AdNT-3) to deliver sustained low doses of NT-3. We assessed its effect in two rat models: streptozotocin (STZ)-induced diabetes, a model of early diabetic neuropathy characterized by demyelination, and acrylamide experimental neuropathy, a model of diffuse axonal neuropathy which, like late-onset human diabetic neuropathy, results in a diffuse sensorimotor neuropathy with dysautonomy. Treatment of STZ-diabetic rats with AdNT-3 partially prevented the slowing of motor and sensory nerve conduction velocities (p < 0.01 and p < 0.0001, respectively). Treatment with AdNT-3 of acrylamide-intoxicated rats prevented the slowing of motor and nerve conduction velocities (p < 0.001 and p < 0.0001, respectively) and the decrease in amplitude of compound muscle potentials (p < 0.0001), an index of denervation. Acrylamide-intoxicated rats treated with NT-3 had higher than control levels of muscle choline acetyltransferase activity (p < 0.05), suggesting greater muscle innervation. In addition, treatment of acrylamide-intoxicated rats with AdNT-3 significantly improved behavioral test results. Treatment with AdNT-3 was well tolerated with minimal muscle inflammation and no detectable general side effects. Therefore, our results suggest that NT-3 delivery by adenovirus-based gene therapy is a promising strategy for the prevention of both early diabetic neuropathy and axonal neuropathies, especially late axonal diabetic neuropathy.

Riluzole prolongs survival and delays muscle strength deterioration in mice with progressive motor neuronopathy (pmn).

The neuroprotective drug riluzole (Rilutek) is a sodium channel blocker and anti-excitotoxic drug which is marketed for the treatment of amyotrophic lateral sclerosis (ALS). Previous studies have shown that riluzole prolongs survival of transgenic mice harboring the mutated form of Cu,Zn-superoxide dismutase found in familial forms of the human disease. In this study we have examined the effect of treatment with riluzole in mice suffering from progressive motor neuronopathy (pmn), a hereditary autosomal recessive wasting disease which shares some symptoms of ALS. These mutants display hind limb weakness starting during the 3rd week of life and leading to paralysis and death during the 7th week of life. Daily treatment with 8 mg/kg of riluzole by oral route significantly retarded the appearance of paralysis, increased life span and improved motor performance on grip test and electromyographic results in the early stage of the disease. There was no effect of riluzole on weight gain. These data demonstrate that riluzole significantly prolongs life span, retards the onset of paralysis and slows the evolution of functional parameters connected with muscle strength in the pmn mouse model of motor neuron disease.

Adenovirus-mediated suicide gene transduction: feasibility in lens epithelium and in prevention of posterior capsule opacification in rabbits.

The most common complication of cataract surgery is the development of posterior capsule opacification (PCO). Hyperplasia of the lens epithelium is one of the main cellular events following phacoemulsification, and has been found to be an important feature contributing to opacification of the posterior capsule. Adenoviral vector-mediated transfer is a suitable method for transducing the herpes simplex virus thymidine kinase gene (HSV-tk) into proliferating cells, allowing for the selective killing of these cells by ganciclovir (GCV) treatment. To determine the potential of gene transduction for lens epithelial cells, we studied the transduction of rabbit lens epithelial cells with adenoviral vectors containing either the Escherichia coli beta-galactosidase (lacZ) gene or the HSV-tk gene in vitro and in vivo in an experimental model of PCO. The efficiency of lacZ gene transfer in rabbit lens epithelial cells was at least 95% both in vitro and in vivo. In vivo transduction with HSV-tk adenoviral vector followed by GCV treatment significantly inhibited the development of PCO (p<0.001). These results suggest that adenoviral vector-mediated transfer of HSV-tk into the proliferating lens epithelial cells is feasible and may provide a novel therapeutic strategy for PCO.

Prominent neuronal-specific tub gene expression in cellular targets of tubby mice mutation.

The tubby strain of mice exhibits maturity-onset obesity and sensory deficits in vision and hearing. The mutated gene, tub , responsible for this phenotype was identified recently, but the function of the TUB protein has not been deduced from its amino acid sequence. This prompted us to undertake expression mapping studies with the hope that they might help to elucidate the biological role of the TUB protein. We report the tub gene expression pattern in embryonic, fetal and adult mice tissues as determined by northern blots and in situ hybridization, using antisense oligonucleotidic probes. In mouse embryos, tub is expressed selectively in differentiating neurons of the ensemble of central and peripheral nervous systems, starting at 9.5 days after conception. In adult mice, tub is transcribed in several major brain areas, including cerebral cortex, hippocampus, several nuclei of the hypothalamus controlling feeding behavior, in the spiral ganglion of the inner ear and in the photoreceptor cells of the retina. These structures contain potential cellular targets of the tubby mutation-induced pathogenesis. The neuronal-specific tub gene distribution allows the establishment of a genotype-phenotype correlation in the tubby mice. This correlation is reminiscent of that observed in fat/fat mice, whose phenotype, also characterized by obesity, is caused by a null mutation in the carboxypeptidase E (CPE) gene. Our observations highlight similarities between CPE, prohormone convertases, several neuropeptides and tub gene expression patterns during embryogenesis, and may narrow down the avenues to explore in order to determine ultimately the function of the TUB protein.

Adenovirus in the brain: recent advances of gene therapy for neurodegenerative diseases.

Adenovirus is an efficient vector for neuronal gene therapy due to its ability to infect post-mitotic cells, its high efficacy of cell transduction and its low pathogenicity. Recombinant adenoviruses encoding for therapeutical agents can be delivered in vivo after direct intracerebral injection into specific brain areas. They can be transported in a retrograde manner from the injection site to the projection cell bodies offering promising applications for the specific targeting of selected neuronal populations not easily accessible by direct injection, such as the motor neurons in the spinal cord. Adenoviral vectors are also efficient tools for the ex vivo gene therapy, that is, the genetical modification of cells prior to their transplantation into the nervous system. Recently, the efficacy of the adenovirus as a gene vector system has been demonstrated in several models of neurodegenerative diseases including Parkinson's disease (PD) and motor neuron diseases. In rat models of PD, adenoviruses encoding for either tyrosine hydroxylase, superoxide dismutase or glial-derived neurotrophic factor improved the survival and the functional efficacy of dopaminergic cells. Similarly, the intramuscular injection of an adenovirus encoding for neurotrophin-3 had substantial therapeutic effects in a mutant mouse model of motor neuron degenerative disease. However, although adenoviruses are highly attractive for neuronal gene transfer, they can trigger a strong inflammatory reaction leading in particular to the destruction of infected cells. The recent development of new generations of adenoviral vectors could shed light on the nature of the immune reaction caused by adenoviral vectors in the brain. The use of these new vectors, combined with that of neurospecific and regulatable promoters, should improve adenovirus gene transfer into the central nervous system.

Adenovirus-mediated gene transfer results in decreased lysosomal storage in brain and total correction in liver of aspartylglucosaminuria (AGU) mouse.

Aspartylglucosaminuria (AGU) is a lysosomal storage disease leading to mental retardation, which is caused by deficiency of aspartylglucosaminidase (AGA). AGU is strongly enriched in the Finnish population in which one major mutation called AGU(Fin) has been identified. The molecular pathogenesis of AGU as well as the biology of the AGA enzyme have been extensively studied, thus giving a profound basis for therapeutic interventions. In this study we have performed adenovirus-mediated gene transfer to the recently produced mouse model of AGU, which exhibits similar pathophysiology as that in humans. Recombinant adenovirus vectors encoding for the human AGA and AGU(Fin) polypeptides were first applied in primary neurons of AGU mouse to demonstrate wild-type and mutant AGA expression in vitro. In vivo, both of the adenovirus vectors were injected into the tail vein of AGU mice and the expression of AGA was demonstrated in the liver. The adenovirus vectors were also injected intraventricularly into the brain of AGU mice resulting in AGA expression in the ependymal cells lining the ventricles and further, diffusion of AGA into the neighbouring neurons. Also, AGA enzyme injected intraventricularly was shown to transfer across the ependymal cell layer. One month after administration of the wild-type Ad-AGA, a total correction of lysosomal storage in the liver and a partial correction in brain tissue surrounding the ventricles was observed. After administration of the Ad-AGU virus the lysosomal storage vacuoles in liver or brain remained unchanged. These data demonstrate that the lysosomal storage in AGU can be biologically corrected and furthermore, in the brain a limited number of transduced cells can distribute AGA enzyme to the surrounding areas.

Intrastriatal injection of an adenoviral vector expressing glial-cell-line-derived neurotrophic factor prevents dopaminergic neuron degeneration and behavioral impairment in a rat model of Parkinson disease.

Glial-cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for adult nigral dopamine neurons in vivo. GDNF has both protective and restorative effects on the nigro-striatal dopaminergic (DA) system in animal models of Parkinson disease. Appropriate administration of this factor is essential for the success of its clinical application. Since it cannot cross the blood-brain barrier, a gene transfer method may be appropriate for delivery of the trophic factor to DA cells. We have constructed a recombinant adenovirus (Ad) encoding GDNF and injected it into rat striatum to make use of its ability to infect neurons and to be retrogradely transported by DA neurons. Ad-GDNF was found to drive production of large amounts of GDNF, as quantified by ELISA. The GDNF produced after gene transfer was biologically active: it increased the survival and differentiation of DA neurons in vitro. To test the efficacy of the Ad-mediated GDNF gene transfer in vivo, we used a progressive lesion model of Parkinson disease. Rats received injections unilaterally into their striatum first of Ad and then 6 days later of 6-hydroxydopamine. We found that mesencephalic nigral dopamine neurons of animals treated with the Ad-GDNF were protected, whereas those of animals treated with the Ad-beta-galactosidase were not. This protection was associated with a difference in motor function: amphetamine-induced turning was much lower in animals that received the Ad-GDNF than in the animals that received Ad-beta-galactosidase. This finding may have implications for the development of a treatment for Parkinson disease based on the use of neurotrophic factors.

In vivo adenovirus-mediated gene transfer to newborn rat retinal pigment epithelial cells.

A successful surgical access to the subretinal space is critical for achieving adenovirus-mediated gene transfer to the retinal pigment epithelial (RPE) cells or photoreceptor cells. We report a novel surgical approach allowing an efficient delivery of recombinant replication-deficient adenoviral vectors into the subretinal space of newborn rats. Our data suggest that this method may be useful for infecting reproducibly large area of the RPE cell layer of normal newborn rats and should be applicable to RCS pups. We also show the feasibility of infecting ex vivo RPE cells in culture using the same recombinant adenoviral vector.

Intrastriatal grafts of embryonic mesencephalic rat neurons genetically modified using an adenovirus encoding human Cu/Zn superoxide dismutase.

Intrastriatal grafting of embryonic dopamine-containing neurons is a promising approach for treating clinical and experimental Parkinson's disease. However, neuropathological analyses of grafted patients and transplanted rats have demonstrated that the survival of grafted dopamine neurons is relatively poor. In the present study, we pursued a strategy of transferring a potentially neuroprotective gene into rat embryonic mesencephalic rat cells in vitro, before grafting them into the denervated striatum of 6-hydroxydopamine-lesioned rats. We performed intrastriatal grafts of embryonic day 14 mesencephalic cells infected with replication-defective adenoviruses bearing either the human copper-zinc superoxide dismutase gene or, as a control, the E. coli lac Z marker gene. The transgenes were expressed in the grafts four days after transplantation and the expression persisted for at least five weeks thereafter. After five weeks postgrafting, there was more extensive functional recovery in the superoxide dismutase group as compared to the control (uninfected cells) and beta-galactosidase groups. The functional recovery was significantly correlated with the number of tyrosine hydroxylase-positive cells in the grafts, although the clear trend to increased survival of the dopamine neurons in the superoxide dismutase grafts did not reach statistical significance. Only a moderate inflammatory reaction was revealed by OX-42 immunostaining in all groups, suggesting that ex vivo gene transfer using adenoviral vectors is a promising method for delivering functional proteins into brain grafts.

Prevention of motoneuron death by adenovirus-mediated neurotrophic factors.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motoneurons, and has no effective treatment. Experimental studies in rodents have shown that motoneurons respond to a variety of molecules including brain-derived neurotrophic factor (BDNF). and the glial-cell line-derived neurotrophic factor (GDNF). Here we investigated the neuroprotective effect of these growth factors, encoded by an adenovirus, on the death of axotomized facial motoneurons in newborn rats. We used a new gene therapy strategy that involves gene transfer to motoneurons by intramuscular injection of an adenoviral vector, which is retrogradely transported from injected target muscle (Finiels et al.,: NeuroReport 7:373-378, 1995). A significant increased survival of motoneurons was observed in animals pretreated with adenovirus encoding BDNF (34.5%, P < 0.05) ou GDNF (41.9%, P < 0.05) 1 week after axotomy. These results indicate that pretreatment with BDNF or GDNF, using this therapeutic strategy, is able to prevent the massive death of motoneurons that normally follows axotomy in the neonatal period, opening new perspectives to limit neuronal death in degenerative disorders.

Immunohistochemical analysis of presenilin 2 expression in the mouse brain: distribution pattern and co-localization with presenilin 1 protein.

Missense mutations of presenilin 1 (PS-1) and presenilin 2 (PS-2) genes cause the majority of early-onset familial forms of Alzheimer's disease (AD). We previously characterized the distribution of the PS-1 protein in the mouse brain by immunohistochemistry using an antibody directed against an epitope located in the large hydrophilic loop [Moussaoui, S., Czech, C., Pradier, L., Blanchard, V., Bonici, B., Gohin, M., Imperato, A. and Revah, F., Immunohistochemical analysis of presenilin 1 expression in the mouse brain, FEBS Lett., 383 (1996) 219-222]. Similarly, we now report the distribution pattern of PS-2 protein in the mouse brain. For these experiments we used a polyclonal antibody raised against a synthetic peptide corresponding to the amino-acid sequence 7-24 of the predicted human PS-2 protein. The specificity of the antibody was evidenced by its ability to recognize PS-2 protein in immunoprecipitation studies and by antigen-peptide competition. In the mouse brain, PS-2 protein was present in numerous cerebral structures, but its distribution in these structures did not correlate with their susceptibility to AD pathology. In all examined structures of the gray matter, PS-2 protein was concentrated in neuronal cell bodies but it was not detected in the glial cells of the white matter. The regional distribution pattern of PS-2 protein was almost identical to that of PS-1 protein. Moreover, PS-2 protein co-localized with PS-1 protein in a large number of neuronal cell bodies. In terms of subcellular localization, PS-2 immunostaining was present almost exclusively in neuronal cell bodies while PS-1 immunostaining was also present in dendrites. This could be explained by the different epitopes of the antibodies and the known proteolytic processing of both presenilins in vivo [Tanzi, R.E., Kovacs, D.M., Kim, T.-W., Moir, R.D., Guenette, S.Y. and Wasco, W., The presenilin genes and their role in early-onset familial Alzheimer's disease, Alzheimer's disease Rev., 1 (1996) 91-98]. Within neuronal cell bodies, the immunostaining of PS-2 protein, as well as that of PS-1 protein, had a reticular and granular appearance. This suggests in agreement with previous observations on PS-1 and PS-2 in COS and H4 cells [Kovacs, D.M., Fausett, H.J., Page, K.J., Kim, T.-W., Moir, R.D., Merriam, D.E., Hollister, R.D., Hallmark, O.G., Mancini, R., Felsenstein, K.M., Hyman, B.T., Tanzi, R.E., Wasco, W., Alzheimer-associated presenilins 1 and 2: neuronal expression in brain and localization to intracellular membranes in mammalian cells, Nature Med., 2 (1996) 224-229] that these proteins are situated in intracytoplasmic organelles, possibly the endoplasmic reticulum and the Golgi complex.

Gene therapy of murine motor neuron disease using adenoviral vectors for neurotrophic factors.

Motor neuron diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy cause progressive paralysis, often leading to premature death. Neurotrophic factors have been suggested as therapeutic agents for motor neuron diseases, but their clinical use as injected recombinant protein was limited by toxicity and/or poor bioavailability. We demonstrate here that adenovirus-mediated gene transfer of neurotrophin-3 (NT-3) can produce substantial therapeutic effects in the mouse mutant pmn (progressive motor neuronopathy). After intramuscular injection of the NT-3 adenoviral vector, pmn mice showed a 50% increase in life span, reduced loss of motor axons and improved neuromuscular function as assessed by electromyography. These results were further improved by coinjecting an adenoviral vector coding for ciliary neurotrophic factor. Therefore, adenovirus-mediated gene transfer of neurotrophic factors offers new prospects for the treatment of motor neuron diseases.

Regional and cellular presenilin 2 (STM2) gene expression in the human brain.

Presenilin 2 (STM2) is a recently cloned gene involved in some forms of early onset Alzheimer's disease with autosomal dominant inheritance. Here we report the regional and cellular distribution of STM2 mRNA in the normal human central nervous system. Using in situ hybridization. STM2 gene expression was shown to be confined exclusively to neurones in the central nervous system. A high level of STM2 mRNA expression was observed in the cerebral cortex and the hippocampus, more particularly on pyramidal neurones of Ammon's horn and granular neurones of the dentate gyrus. STM2 mRNA was also detected in Purkinje cells and granular cells of the cerebellum, and in neurones of the striatum and the nucleus basalis of Meynert. Taken together, these results suggest that the expression of STM2 mRNA is not restricted to the neuronal populations that are known to degenerate in Alzheimer's disease.

Neuromuscular function impairment is not caused by motor neurone loss in FALS mice: an electromyographic study.

Dominant mutations of human Cu/Zn superoxide dismutase (SOD1) are found in about 20% of patients with familial amyotrophic lateral sclerosis (FALS). A transgenic mouse model of FALS (FALSG93A mice) has been generated by overexpression of a mutated form of SOD1. Using electromyography we first show that FALSG93A mice suffer from motoneurone dysfunction similar to that observed in ALS patients and fulfill Lambert's criteria for ALS. We also showed that FALSG93A mice demonstrate a massive loss of functional motor units starting at 47 days of age. Impairment of motor neurone function preceeds by 6 weeks the onset of apparent clinical signs (shaking, tremor) and the beginning of motor neurone loss. Neuromuscular deficits in FALS mice do not result from motoneuronal cell death but rather from loss of axonal integrity.

Immunohistochemical analysis of presenilin-1 expression in the mouse brain.

At least 22 different mutations associated with early-onset familial Alzheimer's disease (AD) in various kindreds have been reported to occur in a recently identified gene on chromosome 14, presenilin 1 (PS-1) (Sherrington et al. (1995) Nature 375, 754-760 [1] and reviewed by Van Broeckhoven (1995) Nat. Genet. 11, 230-231 [2]). In order to study the localization of PS-1 in the brain, we raised a polyclonal antiserum specific to a fragment of the predicted protein sequence of PS-1. PS-1 immunostaining was found intracellularly, in the perikaria of discrete cells, mostly neurons, appearing as thick granules, resembling large-size vesicles. These granules were located in the periphery of cell bodies and extended into dendrites and neurites. PS-1 expression was found to be broadly distributed throughout the mouse brain, not only in structures involved in AD pathology, but also in structures unaltered by this disease.

Direct intracerebral nerve growth factor gene transfer using a recombinant adenovirus: effect on basal forebrain cholinergic neurons during aging.

Gene therapy in the nervous system offers an attractive strategy for the administration of therapeutic factors in a potentially region-specific, sustained, and well-tolerated manner. We tested the trophic effect of a recombinant adenovirus encoding nerve growth factor (AdNGF) in vivo on basal forebrain cholinergic neurons of aged rats, a neuronal population affected during normal and pathological aging. Three weeks after unilateral injection of the recombinant adenovirus into the nucleus basalis magnocellularis, a significant increase in the somal areas of cholinergic neurons ipsilateral to the injection was observed. No increase was detected in animals receiving a recombinant adenovirus carrying the Escherichia coli Lac Z reporter gene. Injected animals did not lose weight, an adverse effect usually described after intracerebroventricular infusion of NGF, and no tissue loss or massive local inflammatory response was observed around injection sites. Thus, a single intracerebral injection of AdNGF produces trophic effects similar to those resulting from chronic intracerebroventricular high levels of NGF. These findings indicate that recombinant adenoviruses encoding growth factors are potentially powerful tools for improving neuronal deficits associated with degenerative processes.

An adenovirus encoding CuZnSOD protects cultured striatal neurones against glutamate toxicity.

Superoxide dismutase (SOD), a key enzyme in the detoxification of free radicals, catalyses the dismutation of superoxide O2.- to oxygen and hydrogen peroxide (H2O2). It is therefore a promising candidate for gene transfer therapy of neurological diseases in which free radicals are thought to be involved. We have constructed a recombinant adenoviral vector containing the human copper-zinc SOD cDNA. Using this vector we were able to drive the production of an active human copper-zinc SOD protein (hCuZnSOD) in various cell lines and primary cultures. Infection of striatal cells with a recombinant adenovirus expressing hCuZnSOD protected these cells from glutamate-induced cell death.

Specific and efficient gene transfer strategy offers new potentialities for the treatment of motor neurone diseases.

Several growth factors are candidates for the therapy of motor neurone diseases. However, there is no efficient, safe, and practicable administration route which hampers the clinical use of these potentially therapeutic agents. We show that specific and high yield gene transfer into motor neurones can be obtained by peripheral intramuscular injections of recombinant adenoviruses. These vectors are retrogradely transported from muscular motor units to motor neurone cell bodies. Gene transfer can thus be specifically targeted to particular regions of the spinal cord by appropriate choice of the injected muscle. The efficiency of gene transfer is high, with 58-100% of the motor neurones afferent to the injected muscle expressing the transgene. This new therapeutic protocol allows specific targeting of motor neurones without lesioning the spinal cord, and should avoid undesirable side effects associated with systemic administration of therapeutic factors.