Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors.

Successful gene therapy approaches will require efficient gene delivery and sustained expression of the transgene in recipients. A variety of methods, ranging from direct DNA delivery to infection with recombinant viruses containing foreign genes, have been developed, but they all have some major limitations that restrict their utility. We have described a human lentiviral (HIV)-based vector that can transduce non-dividing cells in vitro and deliver genes in vivo. With this vector, expression of transgenes in the brain has been detected for more than six months--the longest period tested so far. Because lentiviral vectors are pseudotyped with vesicular stomatitis virus G glycoprotein (VSVG; ref. 8), they can transduce a broad range of tissues and cell types. We now describe the ability of lentiviral vectors to introduce genes directly into liver and muscle. Sustained expression of green fluorescent protein (GFP), used as a surrogate for therapeutic protein, can be observed for more than 22 weeks in the liver. Similar long-term expression (more than eight weeks) was observed in transduced muscle. In contrast, little or no GFP could be detected in liver or muscle transduced with the Moloney murine leukaemia virus (M-MLV), a prototypic retroviral based vector. At a minimum, 3-4% of the total liver tissue was transduced by a single injection of 1-3 x 10(7) infectious units (I.U.) of recombinant HIV vector. Furthermore, no inflammation of recruitment of lymphocytes could be detected at the site of injection. Animals previously transduced with a lentiviral vector can be efficiently re-infected with lentiviral vectors. Additionally, we show that the requirement for lentiviral accessory proteins to establish efficient transduction in vivo is tissue dependent.

In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.

A retroviral vector system based on the human immunodeficiency virus (HIV) was developed that, in contrast to a murine leukemia virus-based counterpart, transduced heterologous sequences into HeLa cells and rat fibroblasts blocked in the cell cycle, as well as into human primary macrophages. Additionally, the HIV vector could mediate stable in vivo gene transfer into terminally differentiated neurons. The ability of HIV-based viral vectors to deliver genes in vivo into nondividing cells could increase the applicability of retroviral vectors in human gene therapy.

Quantitative determination of lentiviral vector particle numbers by real-time PCR.

Here, we describe a quantitative, DNA-based, real-time PCR approach to determine the number of lentivirus particles that are present in vector preparations. In this approach, the minus strong-stop cDNA fragment that is present in viral capsids serves as template for PCR. Using this technology, we found that only 0.1%-1% of the virus particles that are present in vector preparations are infectious. The approach described here is rapid, reliable, and simple in concept and can be used to estimate both vector particles in supernatants and the number of infectious particles. Also, this approach can easily be adapted to a high-throughput system by using 96-well plates and a 2-h running time.

Treatment of refractory pain after brachial plexus avulsion with dorsal root entry zone lesions.

OBJECTIVE: Significant numbers of patients experience intractable pain after brachial plexus root avulsions. Medications and surgical procedures such as amputation of the limb are often not successful in pain treatment. METHODS: Forty-seven patients with intractable pain after traumatic cervical root avulsions were treated with dorsal root entry zone coagulation between 1980 and 1998. The dorsal root entry zone coagulation procedure was performed 4 months to 12 years after the trauma, and patients were monitored for up to 18 years (average follow-up period, 14 yr). RESULTS: Immediately after surgery, 75% of patients experienced significant pain reduction; this value was reduced to 63% during long-term follow-up monitoring. Nine patients experienced major complications, including subdural hematomas (n = 2) and motor weakness of the lower limb (n = 7). Improved coagulation electrodes with thermistors that could produce smaller and more-accurate lesion sizes, which were introduced in 1989, significantly reduced the number of complications. CONCLUSION: Central deafferentation pain that persists and becomes intractable among patients with traumatic cervical root avulsions has been difficult to treat in the past. Long-term follow-up monitoring of patients who underwent the dorsal root entry zone coagulation procedure in the cervical cord indicated that long-lasting satisfactory relief is possible for the majority of individuals, with acceptable morbidity rates.

Shuttle of lentiviral vectors via transplanted cells in vivo.

Lentiviral vectors have turned out to be an efficient method for stable gene transfer in vitro and in vivo. Not only do fields of application include cell marking and tracing following transplantation in vivo, but also the stable delivery of biological active proteins for gene therapy. A variety of cells, however, need immediate transplantation after preparation, for example, to prevent cell death, differentiation or de-differentiation. Although these cells are usually washed several times following lentiviral transduction, there may be the risk of viral vector shuttle via transplanted cells resulting in undesired in vivo transduction of recipient cells. We investigated whether infectious lentiviral particles are transmitted via ex vivo lentivirally transduced cells. To this end, we explored potential viral shuttle via ex vivo lentivirally transduced cardiomyocytes in vitro and following transplantation into the brain and peripheral muscle. We demonstrate that, even after extensive washing, infectious viral vector particles can be detected in cell suspensions. Those lentiviral vector particles were able to transduce target cells in transwell experiments. Moreover, transmitted vector particles stably transduced resident cells of the recipient central nervous system and muscle in vivo. Our results of lentiviral vector shuttle via transduced cardiomyocytes are significant for both ex vivo gene therapy and for lentiviral cell tracing, in particular for investigation of stem cell differentiation in transplantation models and co-cultivation systems.

[Nifedipine versus nitroglycerin in aortocoronary bypass surgery. The effect on hemodynamics, kidney function and homologous blood requirement]. / Nifedipin versus Nitrat bei aorto-koronaren Bypassoperationen. Einfluss auf Hämodynamik, Nierenfunktion und Fremdblutbedarf.

Even during adequate general anesthesia, hypertension is a common phenomenon in patients undergoing aortocoronary bypass grafting (CABG). In such cases application of vasodilators is recommended in order to decrease myocardial oxygen consumption. This study was performed to compare two commonly used substances, i.e., nitrates and nifedipine, with regard to their influence on hemodynamics, renal blood flow, kidney function, and the requirement for homologous blood transfusions. METHODS. Forty-four patients gave their informed consent to the study. They were randomly divided into 2 groups: group 1 received nitroglycerin (3.0 micrograms/kg.min), group 2 nifedipine (Adalat, 0.5 microgram/kg.min) in order to prevent hypertension in the phase before onset of cardiopulmonary bypass (CPB). Anesthesia was induced by etomidate and succinylcholine and maintained as a modified neuroleptanalgesia with fentanyl (up to 50 micrograms/kg), midazolam (0.3 mg/kg.h), and pancuronium (0.1 mg/kg). Systolic blood pressure was kept within the range of 120-160 mm Hg; in case of higher values boluses of either 0.25 mg nitroglycerin or 0.5 mg nifedipine were administered. Cardiac index, stroke volume index, rate-pressure product, intrapulmonary shunt, and pulmonary and total peripheral resistances were evaluated at five predefined points: (1) after induction of anesthesia; (2) before incision; (3) before cannulating the aorta; (4) after decannulating the aorta; and (5) at the end of operation. Creatinine and free-water clearances as well as sodium and potassium excretion were calculated for three phases of the operation: (A) induction of anesthesia--onset of CPB; (B) during CPB; and (C) end of CPB--end of operation. CPB was performed using a membrane oxygenator (Sorin 51) and a nonpulsatile blood flow of 2.5 1/min.m2, which was reduced during mild hypothermia of 30-32 degrees C to 1.7 l/min.m2. Mean arterial pressure in both groups was kept at approximately 70 mm Hg. In case of lower pressures norepinephrine (50-100 micrograms/bolus) was administered; higher pressures were treated as described above. Volume substitution was performed initially by 500 ml hydroxyethyl starch and continued, if necessary, by homologous blood or 5% human albumin in order to keep the hematocrit greater than 30 in the phases before and after CPB. RESULTS. Group 2 showed significantly higher values of cardiac index and stroke volume index at point 3 while the rate-pressure product was clearly lower, indicating better myocardial performance and lower oxygen consumption than in group 1. Creatinine and free-water clearances in all three phases did not differ. However, sodium excretion during CPB was significantly higher in the nifedipine group while potassium excretion showed no differences. The average requirement for blood and blood substitutes was lower in group 2, but the difference could not be confirmed statistically because of the large dispersion of values. Nevertheless, 4 patients in the nifedipine group but no patient in group 1 did not need homologous blood transfusion. CONCLUSION. In comparison to nitrates, nifedipine showed some advantages in the treatment of hypertension during CABG: (1) it provided better myocardial performance; (2) it had a more reliable but not too long-lasting effect on elevated total peripherial resistance, leading to better hemodynamic stability; and (3) by not affecting the capacitance vessels it may necessitate fewer homologous blood transfusions.

Lumbar ependymoma presenting with paraplegia following attempted spinal anaesthesia.

Neurological deterioration from intraspinal haematoma following insertion of a spinal needle is extremely rare. We present the case of a 28-yr-old female, who presented with complete paraplegia following attempted spinal anaesthesia for delivery of her third child. Space-occupying iatrogenic spinal haemorrhage from a previously undiagnosed lumbar ependymoma was found to be the precipitating cause. Following laminotomy with blood clot and tumour removal her neurological function improved.

Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector.

We describe the construction of a safe, replication-defective and efficient lentiviral vector suitable for in vivo gene delivery. The reverse transcription of the vector was found to be a rate-limiting step; therefore, promoting the reaction inside the vector particles before delivery significantly enhanced the efficiency of gene transfer. After injection into the brain of adult rats, sustained long-term expression of the transgene was obtained in the absence of detectable pathology. A high proportion of the neurons in the areas surrounding the injection sites of the vector expressed the transduced beta-galactosidase gene. This pattern was invariant in animals sacrificed several months after a single administration of the vector. Transduction occurs by integration of the vector genome, as it was abolished by a single amino acid substitution in the catalytic site of the integrase protein incorporated in the vector. Development of clinically acceptable derivatives of the lentiviral vector may thus enable the sustained delivery of significant amounts of a therapeutic gene product in a wide variety of somatic tissues.

Applications of gene therapy to the CNS.

Gene therapy is a new method with potential for treating a broad range of acquired and inherited neurologic diseases, where the causative gene defect or deletion has been identified. In addition to gene replacement the application of gene products that reduce cellular dysfunction or death represent new therapeutic options. Gene transfer techniques to express novel proteins using different viral vectors in vitro and in vivo, as well as animal models and human trials will be reviewed in this article. We will focus on a new lentiviral vector as a recent gene transfer method and degenerative disorders of the CNS, and their related model systems.

[Psammomamatoid ossifying fibroma of the frontal sinus with intracranial extension]. / Psammomatoides ossifizierendes Fibrom des Sinus frontalis mit intrakranieller Ausbreitung.

Case report of a 29-year-old woman with an psammomatoid ossifying fibroma of the left frontal sinus. Headache was the presenting clinical symptom. The tumor and its intracranial extension were identified by computed tomography and magnetic resonance tomography. Through a two-step combined neuro-rhinosurgical operation the tumor could be completely removed. Ossifying fibroma is a benign tumor mostly affecting the mandible and maxilla. A more aggressive approach may be more beneficial than expectant observation or curettage in the initial management of this benign neoplasm. Because of the unusual location of this rare entity the case history is published and differential diagnostic and therapeutical implications are discussed.

Primary peripheral T-cell lymphoma of the central nervous system.

UNLABELLED: A 47-year-old man was admitted to our hospital with a nine-week history of visual disturbance, headache, disorientation and facial nerve palsy. Serial cranial magnetic resonance imaging (MRI) revealed progressive bilateral occipital edema with hemorrhage and meningeal involvement. There were no hints of hereditary or acquired immunodeficiency. Laboratory examination for bacterial and viral causes was negative. Open brain biopsy revealed primary central nervous system lymphoma of the extraordinary rare so-called "peripheral" T-cell type. The further course was fatal; the patient died 10 weeks after the onset of symptoms from tumor progression before planned chemotherapy could be started. CONCLUSION: If primary central nervous system lymphoma (PCNSL) is suspected, brain biopsy -- either open biopsy or stereotactic biopsy -- should be performed straight away to enable a rapid start of chemotherapy and/or radiotherapy. Peripheral T-cell lymphoma was highly aggressive in this case leading to the patient's death within several weeks.

Development of a self-inactivating lentivirus vector.

We have constructed a new series of lentivirus vectors based on human immunodeficiency virus type 1 (HIV-1) that can transduce nondividing cells. The U3 region of the 5' long terminal repeat (LTR) in vector constructs was replaced with the cytomegalovirus (CMV) promoter, resulting in Tat-independent transcription but still maintaining high levels of expression. A self-inactivating (SIN) vector was constructed by deleting 133 bp in the U3 region of the 3' LTR, including the TATA box and binding sites for transcription factors Sp1 and NF-kappaB. The deletion is transferred to the 5' LTR after reverse transcription and integration in infected cells, resulting in the transcriptional inactivation of the LTR in the proviruses. SIN viruses can be generated with no significant decreases in titer. Injection of viruses into the rat brain showed that a SIN vector containing the green fluorescent protein gene under the control of the internal CMV promoter transduced neurons as efficiently as a wild-type vector. Interestingly, a wild-type vector without an internal promoter also successfully transduced neurons in the brain, indicating that the HIV-1 LTR promoter is transcriptionally active in neurons even in the absence of Tat. Furthermore, injection of viruses into the subretinal space of the rat eye showed that wild-type vector transduced predominantly retinal pigment epithelium and photoreceptor cells, while SIN vector was able to transduce other types of retinal cells, including bipolar, Müller, horizontal, and amacrine cells. This finding suggests that the HIV-1 LTR can negatively influence the internal CMV promoter in some cell types. SIN HIV vectors should be safer for gene therapy, and they also have broader applicability as a means of high-level gene transfer and expression in nondividing cells.

Bcl-xL protects adult septal cholinergic neurons from axotomized cell death.

Bcl-xL suppresses apoptotic cell death induced by diverse stimuli in cell lines in vitro. To examine the mechanism by which axotomized cholinergic neurons die in vivo, lentiviral vectors expressing Bcl-xL, human nerve growth factor (hNGF), or green fluorescent protein were injected into the septum 3 weeks before transection of the fimbria fornix. Three weeks after transection, Bcl-xL- and hNGF-injected animals showed significantly higher numbers of spared cholinergic neurons compared with control (green fluorescent protein) injected animals. These results provide evidence that adult axotomized cholinergic neurons die of apoptotic death that can be prevented by local delivery of hNGF or intracellular delivery of Bcl-xL.

Highly efficient and sustained gene transfer in adult neurons with a lentivirus vector.

The identification of monogenic and complex genes responsible for neurological disorders requires new approaches for delivering therapeutic protein genes to significant numbers of cells in the central nervous system. A lentivirus-based vector capable of infecting dividing and quiescent cells was investigated in vivo by injecting highly concentrated viral vector stock into the striatum and hippocampus of adult rats. Control brains were injected with a Moloney murine leukemia virus, adenovirus, or adeno-associated virus vector. The volumes of the areas containing transduced cells and the transduced-cell densities were stereologically determined to provide a basis for comparison among different viral vectors and variants of the viral vector stocks. The efficiency of infection by the lentivirus vector was improved by deoxynucleoside triphosphate pretreatment of the vector and was reduced following mutation of integrase and the Vpr-matrix protein complex involved in the nuclear translocation of the preintegration complex. The lentivirus vector system was able to efficiently and stably infect quiescent cells in the primary injection site with transgene expression for over 6 months. Triple labeling showed that 88.7% of striatal cells transduced by the lentivirus vector were terminally differentiated neurons.

[Neuronavigation. Computer-assisted surgery in neurosurgery]. / Neuronavigation. Computergestütztes Operieren in der Neurochirurgie.

Computers in Neurosurgery were limited to diagnostics, planning stereotactic procedures, Radiosurgery and Radiation therapy. The possibility of intra-operative localisation only evolved since the mid 80's with the advent of more powerful computers. The computer adds more precision to microneurosurgical procedures, allowing neuronavigation in interaction with the computer. Diverse neuronavigationsystems are described in the literature [7-9, 11, 13]. We are working with the "ISG Viewing Wand" since april 1994. 113 operations were planed and executed aided by this system. We report on our experiences, the advantages and limitations of this system.

[Intracerebral schwannoma in a child--case report]. / Intracerebrales Schwannom bei einem Kind. Ein Fallbericht.

A case of an intracerebral, intraparenchymatous schwannoma in an 8-year-old child is presented. Clinical, radiological and histological findings as well as possible etiologies are discussed.

Efficient gene transfer into the CNS by lentiviral vectors purified by anion exchange chromatography.

Lentiviral vectors have been shown to stably transduce dividing and non-dividing target cells in vitro and in vivo. However, in vivo gene transfer applications with viral vectors in the central nervous system require highly efficient vector preparations, because only very small volumes can be injected stereotactically without damage to the brain tissue. Since lentiviral vectors are generated in transient co-transfection systems, viral preparations need to be purified and efficiently concentrated before injection into the brain. We describe an alternative procedure to concentrate lentiviral preparations by binding viral particles to an anion exchange column. Viral particles are eluted with sodium chloride, desalted and further concentrated by ultrafiltration. These vector preparations allowed high levels of gene transfer into terminally differentiated neuronal and glial cells and long-term transgene expression without any signs of acute and long-term toxicity or inflammation. The purification of lentiviral vectors from large-scale preparations by anion exchange chromatography allowed us to concentrate the virus to small volumes and to use these preparations to genetically modified target cells in vivo without signs of acute inflammatory responses.

Regeneration of auditory nerve following complete sectioning and intrathecal application of the IN-1 antibody.

The cochlear nerve of adult Lewis rats was following microsurgical exposure in the cerebellopontine angle (CPA). The lesions completely interrupted the auditory nerve axons at the lesion site producing ipsilateral deafness in all animals. The rats were then treated with a recombinant Fab fragment of the antibody IN-1 against nerve growth inhibitory proteins for one to two weeks. An age-matched control group of rats was treated with unspecific mouse IgG antibody. Because the cochlear nerve lesions resulted in significant neuronal apoptosis of spiral ganglion cells, neurotrophin-3 (NT-3) was applied to the lesion site immediately post-injury in some rats. Electrophysiological studies were carried out by recording the brainstem auditory evoked potentials (BAEP) before and immediately after the lesion, and at regular intervals up to 2 months after injury. Cochlear nerve fibres were anterogradely traced by horseradish peroxidase (HRP) or biotinylated dextran amine (BDA) injected into the spiral ganglion. The results achieved in this study were consistent with the following conclusions: 1) transection of the adult rat cochlear nerve at the CPA results in functional deafness, disappearance of BAEP, apoptosis of parent axotomized neurons of the spiral ganglion, and interruption of labelled axons close to the lesion site; 2) NT-3 is able to partially rescue axotomized neurons of the spiral ganglion; 3) injured cochlear nerve fibres show a limited spontaneous sprouting and regrowth response which does not lead to BAEP recovery; 4) intrathecal treatment with IN-1 directed against myelin-associated neurite growth inhibitory proteins promotes significant elongation of the injured fibres; and 5) the regenerating fibres seem to navigate to correct targets, and be able to establish synaptic connections for functional recovery as depicted by BAEP examinations.