discoveries of restriction enzymes by hamilton smith and daniel nathans , and reverse transcriptase by howard temin   and david baltimore   in the 1970s set the stage for an explosion in molecular and clinical genetics .
armed with a sufficient amount of the mrna product of a gene such as the beta globin gene , investigators could now produce a radioactive dna copy ( cdna ) of the mrna and use it as a valuable probe of gene expression .
cloned into an expression plasmid   and transduced into an appropriate cell , the cdna would produce mature beta globin mrna , and cdnas could be manipulated at will in what became the era of recombinant dna technology .
the new discoveries were greeted with enthusiasm by most biologists and clinical investigators but with mounting horror and suspicion by many members of the public and their elected officials , as well as some academics .
frankensteins were thought to be loose in biomedical laboratories ; monsters would be created ; plagues of vicious e. coli would be loosed on an innocent population ; mad scientists would forever contaminate the food supply ; the new genetics would lead to a resurgence of social darwinism .
the specter of nazi medicine roiled some faculty meetings and communities in which the science was rapidly advancing .
rules and restrictions were demanded that would deliberately inhibit the work . to their credit , leaders of this new genetics revolution met in asilomar california , where they established laboratory standards intended to reassure themselves , their colleagues , and the general population that gene manipulation could be rendered safe and useful .
while eager clinical investigators hoped to apply the new genetics in the treatment of inherited diseases , cooler heads recognized that the technology was not sufficiently powerful or predictable .
the more cautious advised the national institutes of health to be very wary of human application because the biological ' rules of the game ' had not yet been established .
that did not stop an american clinical entrepreneur from injecting beta globin cdna into the bone marrows of thalassemic patients in italy and israel .
his reward was failure and opprobrium , and academic medicine encountered a congress increasingly determined to surround clinical research with an ever - growing net of regulation .
how could a cdna be introduced efficiently into a rare , quiescent , mammalian cell , such as a bone marrow stem cell , remain potentially active , and be sufficiently expressed when that stem cell developed into a hematopoietic precursor and subsequently fully differentiated functional blood cell ?
furthermore , could a defective gene be actually replaced in human cells in a targeted fashion by a normal counterpart and still maintain high transcription efficiency , or would such ' plug and socket ' technology be so inefficient that correction would be impossible ?
instead , could cdnas such as a beta globin cdna be carried into the target cell chromosomes on the back of a virus , such as a retrovirus , and could the transduced sequence express its mature mrna regardless of its genomic location ?
the idea of a retrovirus as a gene - transfer agent was first seen as dangerously oncogenic , until 1983 and 1984 when mann , mulligan and baltimore   devised cell lines that would produce replication - defective retroviruses that still exploited the capacity of the viruses to incorporate themselves efficiently in the dna of dividing cells . for the most part ,
the modified retroviral vectors infected human cells at comfortingly low multiplicities of infection . shortly after this
, williams and mulligan   and dick and bernstein , and their colleagues , showed that murine bone marrow cells could be transduced with defective retroviruses carrying cdnas , and that mature nucleated blood cells would carry the foreign cdna for weeks , proving that the murine hematopoietic stem cell , despite its very low rate of division , could be so transduced .
but the percentage of infected cells was very low and expression of the transferred gene was vestigial .
the results suggested that successful gene transfer with cdnas borne on replication - defective retroviruses would require high recombinant viral titer , cell culture systems that would encourage stem cell division without differentiation , and a setting in which target cells would have a selective advantage following gene transfer .
the entire field of gene therapy was energized by the findings of williams and dick and their colleagues , but the barriers to translation and clinical application were soon found to be almost insurmountable .
the slow pace prompted orkin and motulsky   to lower their expectations for immediate clinical application , and focus instead on solving the basic technical and biological problems . in an attempt to gain some clinical traction , blaese and his colleagues   introduced retroviral cdna ex vivo into the mature t cells of patients with immunodeficiency due to mutations in adenosine deaminase .
the treatment provided little if any clinical benefit , and the risk of malignancy was obviously high because the t cells were influenced to divide in culture in order to enhance transduction .
concerns about unwieldy and potentially unsafe clinical research protocols began to mount in the united states .
four levels of review , the recombinant dna advisory committee , individual institutional review boards , individual institutional biosafety committees , and the food and drug administration all established barriers that slowed the pace of gene therapy clinical research to a crawl .
this necessary regulatory environment was onerous enough , but it became even more obstructive when investigators at the university of pennsylvania performed a study of gene replacement in a rare metabolic disorder using adenovirus as a vector in order to infect non - dividing liver cells .
one young adult with the disease died after a high titer of virus was administered .
an investigation revealed that the gene therapists had a financial stake in the company that produced the vector .
that revelation initiated an even higher burden of regulation and added massively to a growing concern about conflict of interest in clinical research - a conflict that continues to roil academic waters to this day .
meanwhile , after four decades of development , the clinical application of allogeneic hematopoietic stem cells in transplantation for the treatment of congenital bone marrow diseases was moving ahead reasonably briskly ( sans gigantic regulatory hurdles ) .
this form of cell - based therapy , though initially applicable in only the 25 - 30% of patients with histocompatible donors , was associated with success in several patients with severe immunodeficiencies , congenital bone marrow failures of several types and even the inherited hemoglobin disorders .
adenovirus vectors were thought to be too immunogenic to be useful but they and their cousin , the tiny adeno - associated virus , were considered to be worthy of evaluation in the transduction of non - dividing cells such as liver cells or endothelial cells .
indeed , high and her co - workers   have made quiet progress in the correction of canine hemophilia with adeno - associated virus .
most groups interested in hematopoietic targets or cancer vaccine protocols continue to focus on defective murine leukemia viruses or lentiviruses .
the latter are thought to have a higher capacity than murine leukemia viruses to integrate within the dna of non - dividing cells .
both the science and the regulatory apparatus seemed to be daunting , and the funding was fragmented and difficult to obtain . but in 2002 cavazzana - calvo and his colleagues   blew new life into the field when they made the startling announcement that they had successfully treated nine of ten patients with x - linked severe combined immunodeficiency ( scid ) , utilizing a fairly standard murine leukemia viral vector that carried the common gamma chain of the interleukin-2 receptor into autologous bone marrow cells .
shortly thereafter , however , cynicism returned when the authors reported that several of the patients had developed t cell leukemia .
careful work by the investigators revealed that the long terminal repeat ( ltr ) of the vector may have a predilection for the lmo2 proto - oncogene on chromosome 11 .
but even random integration of the ltr at the lmo2 site would favor selection of such cells . the lmo2 proto - oncogene is often activated by translocation ( 11:14 ) in t cell leukemia . clearly , the gene - corrected immunocytes had a survival advantage , but the malignant t cells had an even greater survival advantage as well as a growth advantage .
what was once a promising new start for gene therapy became an enormous set - back .
vector safety had always been a pressing issue - now it had become a yawning chasm : of 20 patients with x - linked scid treated by gene transfer in these two trials , 18 are currently alive and with good immune reconstitution , but five have experienced a serious side - effect .
of these five children , one died of therapy - related leukemia , and one died of complications of a subsequent stem cell transplant that was performed as a result of the failure of gene therapy .
have been the results of retroviral correction of oxidase deficiency in chronic granulomatous disease . in two well - described cases ,
correction of granulocyte oxidase deficiency has been achieved but at the cost of clonal proliferation of cells activated at the sites of the mds-1-evi1 , prdm16 or setbp1 proto - oncogenes .
furthermore , other in vitro studies have demonstrated similar insertions by lentiviruses bearing beta globin genes . finally , williams and his co - workers   have temporarily discontinued their pioneering work on the correction of the deficiency of dna repair pathways in hematopoietic stem cells of patients with fanconi anemia , because they are concerned that current vectors that permit rescue of those cells may induce insertional mutagenesis and leukemia in the treated patients , and that focus should be on developing methods of expanding deficient hematopoietic stem cells in this disease . despite these serious setbacks
there have been some recent bright lights . in 2008 , maguire   and bainbridge   and their colleagues reported progress on the treatment of leber 's optic atrophy with an adeno - associated viral vector applied to the retina . and retrovirally transfected epidermal stem cells have been grown into keratinocytes to correct the lesions of epidermolysis bullosum .
more follow - up is needed but the initial results hold promise for local applications of gene transfer .
an encouraging report emerged at a recent annual meeting of the european society of gene and cell therapy .
lentivirus vectors have been used to transduce hematopoietic stem cells in x - linked adrenoleukodystrophy , a progressive demyelinating disease that causes severe debilitation by early teenage years .
long - term and stable gene modification has been observed in 20% of myeloid cells , well within the range to reverse phenotypes in some red cell and myeloid disorders .
finally , auiti and his colleagues have recently reported highly encouraging results in the treatment of adenosine deaminase deficiency .
thus , gene therapy of hematopoietic diseases with retroviruses lumbers in choppy straits twixt the scylla of insufficient gene transfer and the charybdis of leukemia , while the therapy of metabolic and coagulation disorders with adeno- and adeno - associated viruses is blunted by immune reponses to the vectors . to committed gene therapists ,
these are simply the challenges that they have always faced , while those who are engaged in stem cell transplantation , or in finding better halfway measures that support afflicted patients , work as best they can , all the while hoping that the holy grail of gene replacement will one day become a safe reality .
the future could lie in the promising field of site - specific gene correction using modified nucleases , and the conversion of corrected somatic cells such as fibroblasts to functioning hematopoietic stem cells .
cdna : copy dna ; ltr : long terminal repeat ; scid : severe combined immunodeficiency .
the authors are particularly grateful to our colleague david a williams , md , for his contributions to this field and his invaluable assistance during the development of this review .
 though the field has moved with glacial speed , gene therapies have been carried out successfully in patients with bone marrow disorders including immune deficiencies . 
 the field may be poised to move forward more rapidly , but many barriers have yet to be surmounted . 