Gene Therapy (1999) 6 , 994–1005  1999 Stockton Press All rights reserved 0969-7128/99 $12.00 http:/ / www.stockton-press.co.uk/ gt Liposome-mediated NGF gene transfection following neuronal injury: potential therapeutic applications LL Zou 1,2 , L Huang 3 , RL Hayes 4 , C Black 4 , YH Qiu 4 , JR Perez-Polo 5 , W Le 6 , GL Clifton 4 and K Yang 1,2 1 Department of Neurosurgery, 2 Center for Cell and Gene Therapy, 6 Department of Neurology, Baylor College of Medicine, Houston, TX; 3 Department of Pharmacology, University of Pittsburgh, Pittsburgh, PA; 4 Department of Neurosurgery, University of Texas Health Science Center at Houston, Houston, TX; and 5 Department of Human Biochemistry and Genetics, University of Texas Medical Branch at Galveston, Galveston, TX, USA We have systematically investigated the therapeutic poten- ine acetyltransferase (ChAT) activity in cultures following tial of cationic liposome-mediated neurotrophic gene trans- calcium-dependent depolarization injury. In in vivo studies, fer for treatment of CNS injury. Following determination of following intraventricular injections of NGF cDNA com- optimal transfection conditions, we examined the effects of plexed with DC-Chol liposomes, ELISA detected nine- to dimethylaminoethane-carbamoyl-cholesterol (DC-Chol) 12-fold increases of NGF in rat CSF. Further studies liposome-mediated NGF cDNA transfection in injured and showed that liposome/NGF cDNA complexes could attenu- uninjured primary septo-hippocampal cell cultures and rat ate the loss of cholinergic neuronal immunostaining in the brains. In in vitro studies, we detected an increase of NGF rat septum after traumatic brain injury (TBI). Since deficits mRNA in cultures 1 day after transfection. Subsequent in cholinergic neurotransmission are a major consequence ELISA and PC12 cell biological assays confirmed that cul- of TBI, our studies demonstrate for the first time that DC- tured cells secreted soluble active NGF into the media from Chol liposome-mediated NGF gene transfection may have day 2 after gene transfection. Further experiments showed therapeutic potential for treatment of brain injury. that such NGF gene transfection reduced the loss of chol- Keywords: liposomes; NGF; gene therapy; neuronal injury; ChAT; CNS Introduction Investigators studying neuronal injury have long known that administration of neurotrophins could have important therapeutic potential for intervention in the pathological responses to injury and enhancement of functional recovery. 1–10 Recent studies have shown the benefits of administering of exogenous neurotrophic fac- tor to treat traumatic brain injury (TBI). 11,12 Although the administration of exogenous neurotrophic proteins has therapeutic potential, the limitations imposed by protein degradation and the blood–brain barrier could restrict the clinical utility of this approach. Gene transfer is an alter- native means of introducing neurotrophins into the cen- tral nervous system (CNS). During the past few years, significant progress has been made in the development of techniques for transfecting genes into the CNS and exploring their potential to treat CNS disorders. 13–22 Cat- ionic liposomes, which can condense DNA and increase transfection efficiency both in vitro and in vivo, 23–29 have several attractive features as vectors for gene transfer. First, cationic liposomes are non-immunogenic and non- toxic at therapeutic doses. Second, cationic liposomes as DNA carriers can transfect postmitotic, non-dividing cells Correspondence: K Yang, Department of Neurosurgery and Center for Cell and Gene Therapy, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA Received 5 November 1998; accepted 5 February 1999 including neurons. Third, cationic liposomes can be used to deliver multiple genes of any type (linear or supercoiled) nucleic acids. Finally, cationic liposomes are relatively simple to prepare and can be administered into the body by several different routes. Recent advances in the development of more efficient liposome formulations have raised the possibility that liposome-mediated neuro- trophic gene transfection may prove useful for treating CNS injury. 30 Dimethylaminoethane-carbamoyl-cholesterol (DC- Chol) liposomes have a relatively high transfection efficiency and low toxicity. 28 Recently, DC-Chol lipo- somes were approved by the FDA for a clinical trial for cancer immunotherapy. However, no studies have sys- tematically examined whether DC-Chol liposomes could carry neurotrophic genes into brain cells. Neurotrophins, nerve growth factor (NGF) for example, are critical for brain recovery structurally and functionally after injury. In studies regarding traumatically injured brain, the septo-hippocampal cholinergic system is important since it remains a target of damage and dysfunction. 31 Exogen- ous administration with NGF has been shown to spare septal cholinergic neurons from the injury-induced death and degeneration. 1–3 As we know, the CNS is less access- ible to systematic administration of protein and multiple direct injections are inconvenient. It is therefore of special interest to develop an alternative method by which sus- tained and sufficient administration of neurotrophin can be achieved. Our laboratory previously has found that