sign in sign up
<<
Home , Gene therapy, Transfection

(2000) 7, 759–763  2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt NONVIRAL TRANSFER TECHNOLOGY RESEARCH ARTICLE Widespread gene into the central nervous system of primates

Y Hagihara1, Y Saitoh1, Y Kaneda2, E Kohmura1 and T Yoshimine1 1Department of Neurosurgery and 2Division of , Department of Molecular Therapeutics, Osaka University Graduate School of , 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan

We attempted in vivo gene transfection into the central ner- The lacZ gene was highly expressed in the medial temporal vous system (CNS) of non-human primates using the hem- lobe, brainstem, Purkinje cells of cerebellar vermis and agglutinating of Japan (HVJ)-AVE , a newly upper cervical cord (29.0 to 59.4% of neurons). Intrastriatal constructed anionic type liposome with a composition injection of an HVJ-AVE liposome–lacZ complex made a similar to that of HIV envelopes and coated by the fusogenic focal transfection around the injection sites up to 15 mm. We envelope of inactivated HVJ. HVJ-AVE conclude that the infusion of HVJ-AVE liposomes into the containing the lacZ gene were applied intrathecally through cerebrospinal fluid (CSF) space is applicable for widespread the cisterna magna of Japanese macaques. Widespread into the CNS of large animals. Gene Therapy expression was observed mainly in the neurons. (2000) 7, 759–763.

Keywords: central nervous system; primates; gene therapy; HVJ liposome

Introduction ever, its efficiency has not been satisfactory in vivo. Recently HVJ-AVE liposome, a new anionic-type lipo- Despite the promising results in experimental animals, some with the envelope that mimics the human immuno- gene therapy has, so far, not been successful in clinical deficiency virus (HIV), has been developed.13 Based upon 1 situations. As Leiden pointed out, many gene delivery an in vivo trial, this vector has a higher efficiency of gene systems show only limited transgene expression in expression than conventional HVJ liposomes and is humans, unlike those in rodents. Difference in the trans- expected as one of the most useful tools for in vivo gene between humans and rodents might be therapy. We previously tried the intrathecal adminis- responsible for the disparity and thus, more representa- tration of HVJ-AVE liposome-bcl-2 complex in rats, tive conditions of clinical situations are needed to evalu- which successfully resulted in a diffuse gene transfection ate the strategies for gene therapy. The non-human pri- into the entire CNS.14 mate is the most suitable subject for preclinical studies of In the present study, the efficiency of HVJ-AVE lipo- human gene therapy. Therefore, we evaluated the extent somes in the transfer of DNA into monkey CNS was of gene transfection into the central nervous system evaluated by intrathecal injection through the cisterna (CNS) in non-human primate. magna in three Japanese macaques (Nos. 1, 2 and 3). Gene transfection into the CNS using viral and chemi- Widespread gene transfection was seen over the CNS. A cal gene delivery vectors has been investigated for the fourth Japanese macaque (No. 4) received the ‘empty’ purpose of protection against neuronal insults and HVJ-AVE liposome in the same manner to serve as a con- 2 degeneration. with adenovirus (Ad), adeno- trol. These results were compared with those obtained 3 4–6 associated virus (AAV), herpes simplex virus1 or len- by stereotactic injection into the striatum of two Japanese 7,8 tivirus have resulted in the reasonable extent of gene macaques (Nos 5 and 6), which showed focal gene transfection in the target regions of the CNS. expression around the injection site. Both methods Chemical gene delivery reagents including conven- seemed to be useful for CNS gene therapy. tional hemagglutinating virus of Japan (HVJ) liposomes and a cationic liposome–DNA complex have also been developed.9,10 This liposome-mediated gene delivery sys- Results tem has advantages over viral vectors because they can transfer expression cassettes of unlimited size. They do Injection of HVJ-AVE liposome-lacZ into the cisterna not replicate or recombine to form an infectious agent, magna (Table 1, Figure 1) and may not evoke the inflammatory or immune In monkeys 1, 2 and 3, ␤-gal-positive cells were seen in 11,12 responses as reported in adenoviral vectors. How- many parts of the brain. The areas that exhibited the high transfection rates (mean ± s.d.) were: dentate nucleus (59.4 ± 9.1%), Purkinje cells of cerebellar vermis (47.9 ± Correspondence: Y Hagihara 17.8%), trigeminal nucleus (45.6 ± 18.7%), cochlear Received 13 September 1999; accepted 14 January 2000 nucleus (44.1 ± 0.8%), oculomotor nucleus (43.0 ± 7.9%), Primate CNS gene transfection Y Hagihara et al 760 Table 1 Transfection rate while limited gene expression was detected in more dis- tant areas, where the liposome vehicles rarely reached. LacZ (%) In monkey 4, ␤-gal positive cells were absent. In the injection of HVJ-AVE liposomes into the cisterna Frontal cortex 0.67 ± 1.55 magna of monkey CNS, we did not detect any lympho- Caudate nucleus 9.47 ± 5.42 cyte infiltration and neural death. Hippocampus 37.17 ± 1.89 Putamen 2.97 ± 1.16 Parietal cortex 0 Stereotactic injection (Figures 2 and 3) Occipital cortex 0 In monkeys 5 and 6, which received HVJ-AVE liposomes Temporal cortex 0 stereotactically into the striatum, the high lacZ expressing Cerebeller cortex 4.2 ± 2.9 ± ± cells were observed at the injected site (58.5 7.1%; mean Cerebeller vermis 47.87 17.82 ± s.d.). The ␤-gal positive cells were noted in areas more Oculomotor N 43.0 ± 7.93 Dentate nucleus 59.4 ± 9.06 than 10 mm rostral or caudal from the injection tract Cochlear nucleus 44.13 ± 0.81 (Figure 2). High transgene expression was also observed Trigeminal N 45.57 ± 18.73 in the frontal cortex. The ␤-gal-positive cells were rarely Facial nucleus 29.0 ± 2.69 recognized in the temporal, parietal and occipital cortex, ± Olive nucleus 42.13 21.67 which were farther than 15 mm from the tract. Choroid plexus 100 Despite the high transgene expression in the frontal ␤ cortex around the needle insertion, gene expression in the Labeling index of -gal in the CNS after an intrathecal injection of striatum was lower than expected (9.5 ± 5.4%; mean ± HVJ-AVE liposome-lacZ (mean ± s.d.%, n = 5). s.d.). The small leakage of the liposome mixture seen at the injection site might have been responsible for this lim- olive nucleus (42.1 ± 21.7%), hippocampus (37.2 ± 1.9%) ited extent of expression. and facial nucleus (29.0 ± 2.7%). The following areas exhi- bited only limited or no transgene expression: caudate Immunohistochemistry (Figure 4) nucleus (9.5 ± 5.4%), cerebellar cortex (4.2 ± 2.9%), Approximately 90 ± 6% (mean ± s.d.) of the ␤-gal-positive putamen (3.0 ± 1.2%), frontal cortex (0.6 ± 1.6%), parietal cells were stained with anti-neurofilament , cortex and occipital cortex (0%). High transgene which are considered as terminally differentiated neu- expression was observed in the regions close to the cis- rons. We could not identify any GFAP-positive cells terna magna, where the HVJ-AVE liposome was applied, among cells exhibiting blue staining in the presence of

a d g

b e h

c f i

Figure 1 ␤-Gal expression in the brain after an intrathecal injection of HVJ-AVE liposome-lacZ complex. Broad ␤-gal expression was seen with X-gal staining (a, b, d, e, g and h). No ␤-gal expression was seen in the frontal cortex (i). The intrathecal injection of HVJ-AVE liposome containing no DNA resulted in no ␤-gal-positive cells (c, f). (a) Cerebellar vermis. Blue stained Purkinje cells were seen among the granular cell layer (×10). (b) Dentate nucleus (×20). (c) Cerebellar vermis, control study (administration with HVJ-AVE liposome carrying no DNA). (d) Hippocampus (×4). (e) Hippocampus (×40). (f) Hippocampus, control study (administration of HVJ-AVE liposome carrying no DNA (×4). (g) Upper cervical cord (×4). (h) Upper cervical cord (×10). (i) Frontal cortex (×4).

Gene Therapy Primate CNS gene transfection Y Hagihara et al a 761

a d

b e

b

c f

Figure 2 Stereotactic injection of HVJ-AVE liposome–lacZ complex into the striatum assayed with X-gal (a–f). (a) Dense ␤-gal expression at the cortical incision (×4). (b) Refluxed HVJ-AVE liposome–lacZ was diffused and transfected into the cortex from the brain surface (×20). (c) ␤-Gal- positive cells were seen in the deep cortical neurons, which suggests that the HVJ-AVE liposome penetrated into the cortex (×4). (d) High ␤-gal expression at the injection tract (×4). T: injection tract. (e) High ␤-gal expression at the injection site (×20). (f) Diffusion of ␤-gal expressing cells in the white mater (×4). *: injection site.

Figure 4 Immunohistochemistry. Staining with anti-neurofilament anti- body (a ×40) and anti-GFAP antibody (c ×10). Most of the ␤-gal express- ing cells were positively stained with anti-neurofilament antibody. None of the ␤-gal expressing cells were positive for anti-GFAP antibody staining.

The same procedure with HVJ-cationic liposome was examined by Mabuchi et al15 in an experimental mouse with glioma. Only disseminated glioma cells in the cere- brospinal fluid space expressed the lacZ gene. LacZ trans- fection was absent in either the neuronal or glial cells. This difference between HVJ-AVE and HVJ-cationic lipo- somes may result from the inability of cationic liposomes to penetrate tissues because of both net positive charge and large size (approximately 700 nm in diameter).13 In Figure 3 Transfection efficiency by the distance from the injection site. contrast, anionic type liposomes can penetrate into tissues, and have higher transfection efficiency in vivo. X-gal. The types of remaining ␤-gal-positive cells were For the purpose of gene transfection into the CNS, some not identified. researchers4–6 have used of defective her- pes simplex virus into the rat CNS, but these attempts Discussion resulted in the gene expression in only a limited region adjacent to the injection sites. A detailed examination of In the present study, widespread transfection of the lacZ the efficiency of transfection into the target cells has not gene into monkey CNS was achieved using the HVJ-AVE been reported. Many researchers also used Ad and AAV. liposome by intrathecal injection through the cisterna Ad is thought as one of the suitable gene vectors for in magna. A high efficiency of gene transfection was seen vivo delivery into the CNS, because it can be applied in in the medial temporal lobe, brainstem and cervical cord. high titer and delivers efficiently to the nonrep- On the other hand, the injection of HVJ-AVE liposome licating and replicating cells. However, its major limi- into the striatum caused high transfection efficiency only tations are its and cytotoxicity. It is in the frontal lobe and striatum. known that Ad evokes nonspecific inflammation and

Gene Therapy Primate CNS gene transfection Y Hagihara et al 762 antivector cellular immunity.12 AAV causes less immuno- DNA, conjugated with UV-inactivated HVJ and adjusted genicity, but its efficiency of in vivo CNS transfection is in 1.0 ml of BSS for intrathecal injections and 100 ␮lof significantly lower than that of Ad and other viral BSS for intrastriatal injections. vectors.7 -based retroviral vectors have higher trans- DNA fection efficiency and sustained gene expression than pcDNA3 (, Leek, The Netherlands), containing AAV, Ad and Moloney virus. Blo¨mer et al7 the (CMV) promoter (655 bp), was evaluated in vivo gene transfer by microinjection into the restricted with HindIII–BamHI. The Escherichia coli ␤-gal striatum of rat CNS and found that the transgene cells gene, isolated from the pSV-␤-gal vector (Promega, Madi- spread only 3 to 4 mm from the injection site. Most of the son, WI, USA) by restriction with HindIII–BamHI and transfected cells (88.7%) were identified as differentiated contains the SV40 nuclear transport signal in its N-ter- neurons. The gene expression was sustained for at least minus, was cloned into the restricted pcDNA3 24 weeks. (CMVlacZ). Transfection with HVJ-AVE liposome produced ␤-gal- positive cells as far as 15 mm rostral and caudal to the In vivo gene transfer injection site. We did not evaluate the stability of gene Monkeys 1, 2 and 3 were anesthetized with pentobarbital expression. However, there is evidence that transfected sodium (50 mg/kg). HVJ-AVE liposome containing the

gene expression was sustained for at least 2 weeks in the lacZ gene in BSS (1.0 ml) with 1 mm CaCl2 was injected mouse liver and for 1 month in mouse skeletal muscle.13 into the cisterna magna of the monkeys using a 1 ml syr- Therefore, the HVJ-AVE liposome seems a promising inge with a 23-gauge needle. Monkey 4 received 1.0 ml new vector for the transfection of neurons into the CNS. of HVJ-AVE liposome containing no DNA using the The reason why the HVJ-AVE liposome delivers genes same procedure. into neuronal cells and not glial cells is still unclear. Len- The other monkeys received HVJ-AVE liposome stere- tivirus vectors, which are also based on the HIV virus, otactically into their striatum (monkeys 5 and 6). Each exhibit highly specific transfection into adult neurons. monkey was placed and fixed in a stereotactic apparatus, Blo¨mer et al explained this specificity by the presence of and a burr hole was made in the skull at a point 18 mm vesicular stomatitis virus (VSV)-G in the lenti- anterior to the external meatus and 9 mm lateral to the virus envelope and the enhancer domain of the hCMV left of the midline. The dura mater was carefully cut in promoter in the neuron. The VSV-G protein can broaden a cruciate manner and the pia mater was coagulated the host cell specificity because it can be recognized by under the operating microscope. The needle was inserted the G protein receptors widely expressed on the cell sur- vertically 22 mm from the brain surface, and HVJ-AVE face while the hCMV major immediate–early promoter liposome containing lacZ (100 ␮l) was injected at a speed (HCMV MIEP) expressed in neurons drives the of 5 ␮l/min into the left striatum. No specific symptoms, expression of the transfected gene preferentially.7,16 These including weight loss or loss of activity, were seen in any hypotheses may support, at least partially, our results of of the monkeys after administration of the HVJ-AVE neuron-specific transfection. Our lacZ gene is under the liposome. control of the hCMV promoter and may exhibit preferen- tial expression in neurons. Although HVJ does not pos- Histologic examination sess the VSV-G protein in its envelope, the HN protein, Seven days after transfection, the monkeys were anes- which is present in the HVJ envelope, can bind to recep- thetized with an overdose of ketamine and pentobarbital tors in most mammalian cells. The envelope protein and sodium, and perfused with 4% paraformaldehyde and the HCMV MIEP may also partly explain the specificity 0.1% glutaraldehyde in phosphate-buffered saline (PBS) of the gene transfection of the HVJ-AVE liposome. (500 ml/kg). The brains were removed and placed in 30% This is the first study demonstrating extensive gene sucrose in PBS for several days. After the meninges were transfection in almost entire CNS of non-human pri- carefully removed under the stereomicroscope, 40-␮m- mates. Our final goal is to establish a therapeutic strategy thick frozen serial sections were made in the frontal for brain protection in humans. Further studies will be plane. focused on the function of the transfected gene, for To detect transgene ␤-gal expression in the brain, the example, anti-apoptotic gene over the ischemic brain. sliced brain sections were rinsed three times with PBS, and stained in X-gal buffer (5-bromo-4-chloro-3-indolyl- ␤ d m Materials and methods - -galactopyranoside (X-gal, 0.5 mg/ml), 3 m K3Fe (CN)6,3mm K4Fe (CN)6 3H2O, 1 mm MgCl2,10mm KCl, 0.1% Triton X-100 in 0.1 m sodium phosphate buffer, pH Animals 7.5) at 37°C for 8 h and counterstained with hematoxylin. Six adult female Japanese macaques, ranging in age from The efficiency of gene transfection was evaluated by 5 to 10 years and weighing from 4.75 to 7.80 kg, were counting the blue staining cells in five random high used in the study. They were provided with a standard power (×100) fields of five slides in each part of the CNS diet of monkey biscuits supplemented with fresh fruit. of monkey 1, 2 and 3 (intrathecal injection). The averaged percentages of transgene expression were calculated by HVJ-AVE liposome counting blue staining cells in 1000 hematoxylin positive The detailed preparation of HVJ-AVE liposomes has been cells in each part of the CNS per monkey. described previously by Saeki et al.13 Briefly, each lipid The character of the transfected cells was determined (9.75 mg of ) mixture was hydrated in 200 ␮lofa by immunohistochemistry using neurofilament as a balanced salt solution (BSS: 137 mm NaCl, 5.4 mm KCl, marker for neuronal cells and glial fibrillary acidic pro- 10 mm Tris-HCl, pH 7.6) containing 100 ␮g of plasmid tein (GFAP) as a marker for astrocytes. X-gal-stained

Gene Therapy Primate CNS gene transfection Y Hagihara et al 763 slices were treated with 0.3% hydrogen peroxidase in 3 Mandel RJ, Spratt SK, Snyder RO. Midbrain injection of recom- methanol for 20 min to inactivate any endogenous per- binant adeno-associated virus encoding rat glial cell line-derived oxidase. They were then incubated with 1% normal horse neurotrophic factor protects nigral neurons in a progressive 6- and goat serum for 10 min, followed by an overnight hydroxydopamine-induced degeneration model of Parkinson’s disease in rat. Proc Natl Acad Sci USA 1997; 94: 14083–14088. incubation at 4°C with monoclonal anti-neurofilament 4 Linnik MD, Zahos P, Geschwind MD. Expression of bcl-2 from antibody (SMI 32; Sternberger Monoclonals, Lutherville, a defective -1 vector limits neuronal death MD, USA) and polyclonal anti-GFAP antibody (PC10555; in focal cerebral ischemia. Stroke 1995; 26: 1670–1675. Progen Biotechnik, Heidelberg, Germany). The sections 5 Lawrence MS, Ho DY, Sun GH. Overexpression of bcl-2 with were washed three times in PBS, incubated with a second herpes simplex virus vectors protects CNS neurons against antibody (1:400; Vector Laboratories, Burlingame, CA, neurological insults in vitro and in vivo. J Neurosci 1996; 15: USA) for 1 h, and then incubated for 1 h with avidin- 486–496. biotin-horseradish peroxidase complex (Vector 6 Lawrence MS, McLaughlin JR, Sun G-H. Herpes simplex viral Laboratories). The histochemical localization of HRP was vectors expressing bcl-2 are neuroprotective when delivered visualized with 3,3Ј-diaminobenzidine (DAB) and hydro- after a stroke. J Cereb Blood Flow Metab 1997; 7: 740–744. 7 Blo¨mer U, Naldini L, Kafri T. Highly efficient and sustained gen peroxide. Ten slides were collected around the injec- gene transfer in adult neurons with a lentivirus vector. J Virol tion site of CNS of monkey 5 and 6 (stereotactic injection) 1997; 71: 6641–6649. and the percentage of double staining cells with X-gal 8 Naldini L, Blo¨mer U, Gage FH. Efficient transfer, integration, and antineurofilament antibody or anti-GFAP antibody and sustained long-term expression of the transgene in adult rat were calculated by counting 1000 blue staining cells per brains injected with a lentiviral vector. Proc Natl Acad Sci USA monkey. 1996; 93: 11382–11388. All animal care and experimental procedures were in 9 Yamada Y, Moriguchi A, Morishita R. Efficient accordance with the NIH Guide for the Care and Use of delivery using the HVJ-liposome method in the central nervous Laboratory Animals (1996) and approved by the Animal system. Am J Physiol 1996; 271: R1212–R1220. Experiment Committee of Graduate School of Medicine, 10 Kaneda Y et al. The improved efficient method for introducing macromolecules into cells using HVJ (Sendai virus). Exp Cell Res Osaka University. 1987; 173: 56–69. 11 Hirano T et al. Persistent gene expression in rat liver in vivo by repetitive using HVJ-liposome. Gene Therapy 1998; Acknowledgements 5: 459–464. 12 Crystal RG. Transfer of genes to humans: early lessons and We greatly appreciate the technical support of Ichiro obstacles to success. Science 1995; 270: 404–410. Fujita, Hiroyuki Yoneshima, Hisashi Tanigawa and Kei- 13 Saeki Y et al. Developement and characterization of cationic lipo- suke Kawasaki, Laboratory of Cognitive Neuroscience, somes conjugated with HVJ (Sendai virus): reciprocal effect of Department of Biophysical engineering, Osaka Univer- cationic lipid for in vitro and in vivo gene transfer. Hum Gene sity Graduate School of Engineering Science. Ther 1997; 8: 2133–2141. 14 Hagihara Y, Saitoh Y, Kaneda Y. Brain protection by the trans- fection of bcl-2 gene into the entire central nervous system References (CNS). Rest Neurol Neurosci 1998; 13: 221–235. 15 Mabuchi E et al. Gene delivery by HVJ-liposome in the experi- 1 Leiden JM. Gene therapy – promise, pitfalls, and prognosis. New mental gene therapy of murine glioma. Gene Therapy 1997; 4: Engl J Med 1995; 13: 871–873. 768–772. 2 Wang C, Chao C, Maddedu P. Central delivery of human tissue 16 Baskar JF et al. The enhancer domain of the human cytomegalo- kallikrein gene reduces blood pressure in hypertensive rats. virus major immediate–early promoter determines cell type spe- Biochem Biophys Res Commun 1998; 244: 449–454. cific expression in transgenic mice. J Virol 1996; 70: 3207–3214.

Gene Therapy