GTMB 7 - Gene Therapy & Molecular Biology

Gene Therapy and Molecular Biology Vol 7, page 189carrying the transgene inserted at 11p13 in the region ofLMO2, an oncogene frequently overexpressed in T cellleukemias (Marshall 2002). Insertional mutagenesis is anacknowleged potential complication with retroviralmediated gene transfer because gene integration occursrandomly into the genome. This is the first report ofmalignant change in humans following retroviral genetherapy and only one example has been found in extensiveanimal studies using this vector (Li et al, 2002).Investigations are ongoing to determine whether any otherfactor contributed to the development of insertionalmutagenesis and clonal expansion in these particularpatients (Friedmann 2003).2. LentivirusBecause of the limitation of infection to dividingcells by retroviruses, alternative vectors such aslentiviruses have been developed to circumvent thisrestriction. Significant progress has been made in recentyears in the development of lentiviral vectors, a retroviralsub-group based on the Human Immunodeficiency Virus(HIV) (Trono, 2000) or Equine Infectious Anaemia Virus(EIAV) (Mitrophanous et al, 1999). HIV vectors arecapable of transferring genes into nondividing cells suchas neurons (Naldini et al, 1996) and quiescenthaematopoietic progenitor cells, (Case et al, 1999) whichwill be particularly useful for these tissue targets.Lentiviral vectors integrate into the genome randomly andare therefore theoretically able to cause insertionalmutagenesis.Lentiviruses can be made more stable bypseudotyping which allows virus titres to be improved byultracentrifugation. This offers the opportunity of infectinga greater number of cells in vivo and different envelopesallow targeted gene transfer to specific tissues, forexample to the nervous system (Mazarakis et al, 2001) andairways (Kobinger et al, 2001). Both the EIAV vector, avector derived from non-primate animal lentiviruses,(Mitrophanous et al, 1999) and Feline ImmunodeficiencyVirus (FIV) (Wang, et al, 1999) have been developed in anattempt to create vectors for use in human treatment whichare not associated with any human pathology. Our recentwork has shown that high level sustained transgeneexpression can be achieved in a variety of tissues using theEAIV vector in fetal mice after intravascularadministration (Figure 1) (Waddington et al, 2003).3. Adeno-associated viral vectorsAdeno-associated virus (AAV) is also a promisingnovel vector system. It is a common human parvovirusthat is not associated with any human pathology. AAVnaturally requires co-infection with adenovirus as a helpervirus, but the latest AAV vectors circumvent the need foradenovirus and therefore make the production of pureAAV particles easier (Xiao et al, 1998). AAV is also ableto infect non-dividing cells and to achieve long-lastinggene correction in vitro and in vivo (Herzog et al, 1999;Wang et al, 1999; Kay et al, 2000). The basis for longtermtransgene expression is not quite clear. Integration ofthe wild type virus is predominantly at an apparentlyspecific functionally unimportant location on humanchromosome 19 reducing the theoretical risk of insertionalmutagenesis; however recombinant vector appears tointegrate at low levels and non-specifically (Monahan andSamulski, 2000). AAV vectors have a limited capacity forthe insertion of foreign genes that is about 4.7kb, althoughrecently 'split AAV vectors' have been designed wherelarge genes are split between two AAV genomes toincrease AAV packaging capacity. Afterconcatemerisation of these genomes in the host cellmRNA, splicing allows the removal of intervening ITRsequences and restoration of the split coding sequence toyield wild-type functional protein (Sun et al, 2000).Because the extent of AAV integration is still in question,this vector system may not give the permanent geneexpression ideal for in utero gene therapy without repeattreatment, although long term transgene expression afterintraperitoneal delivery in mice has recently been reported(Lipshutz et al, 2003). Some caution has also beenexpressed as AAV integration appears to inducechromosome deletions (Nakai et al, 2003).4. AdenovirusAdenoviral vectors have been used as attractivevectors for proof of principle studies in fetal gene therapysince they have continually achieved highly efficient genetransfer in vivo. The adenoviral coding sequencesnecessary for viral replication are deleted, rendering themreplication defective. They are relatively stable and can beobtained at high titre making systemic administration inhumans and large animal models feasible. The adenovirusgenome replicates outside the chromosome, which avoidsthe risk of insertional mutagenesis but results in onlytransient gene expression. Their broad host range andtropism to most cells of the human body, including therespiratory epithelium has made them very useful in initialpathfinder studies on vector delivery and transgeneexpression. They are particularly useful for exploringdifferent technical approaches to fetal gene therapy.Factors that determine the kinetics of transgeneexpression include vector elimination, since adenovirus isnot an integrating vector, and promoter shutdown.Adenoviral vectors are also highly immunogenic. Majorconcerns about the safety of adenoviral vectors wereraised following the death of Jesse Gelsinger from asystemic inflammatory response to a first generationadenovirus vector used for a phase I clinical trial towardsgene therapy of the inherited metabolic disorder, ornithinetranscarbamylase deficiency (Lehrman, 1999). Even fetaladministration of adenoviral vectors has been associatedwith an immune response (McCray, et al, 1995)particularly after postnatal repeat exposure to the vector(Iwamoto et al, 1999). Attempts to reduce theimmunogenicity and toxicity of the vector and to increaseits insert capacity have led to the generation of the socalled ‘gutless vectors’ in which essentially all adenoviralcoding sequences have been eliminated (Chen et al, 1997;Schiedner, et al, 1998).189