A group of scientists led by William M.Pardridge, Professor of Medicine at theUniversity of California, Los Angeles(UCLA; http://www.medsch.ucla.edu),have found that pegylatedimmunoliposomes (PIL) can be usedto transfer genes from the blood intothe brain of primates. Their results,which also demonstrate the exogenousgene is expressed at high levels, buildon earlier studies in rodents andsuggest that this approach might beapplicable in humans.

The delivery problemGene therapy holds tremendouspromise for the treatment of manyhuman diseases. However, the field hasundergone significant setbacks becauseof the potentially lethal side effects ofthe viral vectors used to deliver genes.An adenovirus vector used in a clinicaltrial at the University of Pennsylvania isbelieved to have caused the death of18-year-old Jesse Gelsinger in 1999(http://www.med.upenn.edu). Morerecently, the US Food and DrugAdministration (http://www.fda.gov)placed all active trials using retroviralvectors to insert genes into blood stemcells on ‘clinical hold’ after news that asecond child treated for X-linkedsevere combined immunodeficiencydisease in France had developed aleukemia-like condition.

Pardridge’s team avoids this problemby using PILs as a delivery system. This so-called ‘artificial virus’encapsulates the therapeutic geneinside a liposome, which is then coatedwith polyetheylene glycol (PEG). The liposome prevents degradation ofthe gene by endonucleases, and the pegylation process reduces theabsorption of serum proteins to the

liposome surface, which minimizes theiruptake by cells lining the liver andspleen (the reticulo-endothelial system).

The final step is to target the PIL, inthese experiments to neurons, byattaching a monoclonal antibody, to1–2% of the PEG strands, thatrecognizes receptors expressed on boththe blood–brain barrier (BBB) and thebrain cell plasma membrane (BCM).Pardridge refers to these targetingmonoclonal antibodies as a ‘molecularTrojan horse’. He explains, ‘It enablesthe PIL to first cross the blood–brainbarrier and subsequently to cross theplasma membrane of the neuron. Onceinside the neuron, it moves across thenuclear membrane, where the gene isexpressed.’ How this last step occurs is not understood.

Blood–brain barrierThe approach used by the UCLAresearchers is a novel way to get drugs

through the BBB. ‘This is a monumentalproblem for drug development because98% of all small-molecule drugs and100% of all large molecule drugs,including recombinant proteins,antisense RNA and gene medicines, do not cross the blood–brain barrier,’said Pardridge.

In their most recent study, a singleintravenous injection of PIL, targeted tothe brain with a mouse monoclonalantibody to the human insulin receptor,led to global expression of anexogenous gene coding for bacterialβ-galactosidase in the adult rhesusmonkey brain within 48 hours (Fig. 1).Furthermore, they showed thatexpression of an exogenous geneencoding luciferase was 50-fold higherin the rhesus monkey than seen in therat, which used PIL targeted to thebrain with a mouse monoclonalantibody to the rat transferrin receptor[2]. ‘This particular work is the mostinnovative and potentially the mostground breaking that anybody inblood–brain research has done,’ saidEain Cornford, Professor of Neurologyat UCLA. ‘It could well revolutionizethe field.’

Furthermore, most inherited diseasesthat affect the brain result from defectsin metabolic pathway genes. ‘Theability to get the gene in cells throughthe brain is important to theseprocesses,’ added John Wolfe, Professorof Pathology and Medical Genetics atthe University of Pennsylvania. ‘This[research] is an important step towardsgetting widespread delivery.’

Although expression of the twoexogenous genes also occurred in other organs, such as the liver andspleen, Pardridge has shown that thiscan be eliminated in mice through the

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Genes go incognito into brainVida Foubister, freelance writer

Figure 1. A section of adult rhesusmonkey brain showing globalexpression of an exogenous bacterialβ-galactosidase gene. The bacterialgene was encapsulated in pegylatedimmunoliposomes (PIL) that target the human insulin receptor anddelivered by a single intravenousinjection. Reproduced, withpermission, from Ref. [1].

use of a brain-specific promoter [6]. In the primate experiments, theexogenous genes were on plasmidsunder the influence of a widelyexpressed SV40 promoter.

However, the fact that the exogenousgenes do not integrate into the genome,and thus their expression is short lived,could limit the therapeutic applicationsof this synthetic vector. ‘If you’re tryingto treat an inherited disease, you’relooking for permanent expression,’ saidWolfe. Transient expression might bepreferable for some diseases, and it alsomight be possible to deliver the genesusing a vector that results inpermanent expression, he added.

Promise in ratsPardridge’s team has alreadydemonstrated they can use this

delivery system to get a pharmacologicaleffect in rodents. A plasmid thatexpresses antisense mRNA to thehuman epidermal growth factorreceptor injected intravenously in a PILled to 100% increase of survival time inmice with brain cancer [3]. In addition,the effect of a neurotoxin that causesParkinson’s like symptoms in rats wasreversed by using PIL to deliver theaffected enzyme, tyrosine hydroxylase[4]. ‘We showed that gene therapy notonly normalizes the biochemistry butalso eliminates the motor abnormalityof the disease.’

The team is currently working todevelop a brain-specific promoter forParkinson’s disease and also to use anRNA interference-based approach inthe mouse brain cancer model: thecombination of gene therapy and

RNAi removes aberrant expression ofthe gene that is specific to the cancer,however, according to Pardridge, theproblems preventing this fromhappening are ‘delivery, delivery and delivery!’

References1 Zhang, Y. et al. (2003) Global non-viral gene

transfer to the primate brain followingintravenous administration. Mol. Ther. 7,11–18

2 Shi, N. et al. (2001) Brain-specific expressionof an exogenous gene following intravenousadministration. Proc. Natl. Acad. Sci. U. S. A.98, 12754–12759

3 Zhang, Y. et al. (2002) Antisense genetherapy of brain cancer with an artificialvirus gene delivery system. Mol. Ther. 6,67–72

4 Zhang, Y. et al. (2003) Intravenous non-viralgene therapy causes normalization of striataltyrosine hydroxylase and reversal of motorimpairment in experimental Parkinsonism.Hum. Gene Ther. 14, 1–12

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Gene therapy success for Alzheimer’s?Matt Brown, m.brown@elsevier.com

Using gene therapy, the levels of aprotein implicated in Alzheimer’sdisease have been dramatically reducedin mice. Researchers from the SalkInstitute (http://www.salk.edu) and theUniversity of California, San Diego(UCSD; http://www.ucsd.edu) haveintroduced the gene for neprilysin intothe brains of transgenic mice, reducinglevels of amyloid protein by up to 50% [1]. Their findings offer hope tothe millions of Alzheimer’s sufferers, and their families, around the world.

More common than you might thinkAlzheimer’s disease (AD) is the mostcommon form of neurodegenerativedisease in people over 65 years. It isestimated that >15 million people are

affected by the disease worldwide andalmost half of those over 85 years showsigns of the disease [2]. The cognitiveareas of the brain are the first to beaffected, leading, amongst other things,to memory loss and behaviouralabnormalities. The disease then spreadsto the parts of the brain that controlmovement, and the patient requiresconstant care. Eventually, the loss ofbrain function becomes so severe that it can be the primary cause of death. As the average lifespan of people in the Western world increases, so toowill the numbers of people affected byneurodegenerative illnesses. Needlessto say, a tremendous amount of time,effort and money has been pouredinto finding a means to cure orprevent the disease.

A complicated aetiologyDespite great strides in AD research,the precise mechanisms that lead tothe disease are not fully understoodand many genetic, cellular andmolecular irregularities are implicated.One of the most established factors,however, is a build-up of amyloidproteins around brain cells to formharmful plaques. Mutations in theamyloid-precursor protein (APP) genecan result in increased production ofAβ1–42, the form of amyloid proteinthat clusters into the harmful plaques.However, in rarer forms of AD, otherfactors might be contributory. Forexample, amyloid build up might becaused by a decreased Aβ1–42 clearance,or from a shift in the balance ofproduction and degradation.