La Enseñanza del Emprendimiento como Proceso de Subjetivación en el Contexto Neoliberal

Since their discovery, retroviruses have been implicated in carcinogenesis

[158, 159]. Indeed studies of tumor-associated retroviruses have contributed to our

understanding of the development of cancer [6]. Retroviruses can exert oncogenic

effects by encoding an oncogene, either a captured cellular gene [159-161] or a

modified viral factor with oncogenic properties [162]. Alternatively, retroviruses

close to the site of proviral integration by insertional activation.

Insertional activation can be caused by a number of mechanisms [6]. One is

upregulation of transcription of an oncogene by retroviral promoter or enhancer

sequences inserted a short distance upstream of the gene. Alternatively, proviruses

may integrate within a gene, resulting in transcriptional readthrough, forming a

hybrid transcript of viral and host sequence. This hybrid may act as an aberrantly

active growth factor, for example encode a constitutively active oncogene missing a

regulatory domain. Finally, an integrated provirus may separate a growth-control

gene from non-coding regions that modulate its expression. Tumors arising by

insertional activation usually have a long latency – assuming the altered locus has a

dominant phenotype, the initial integration event may impart a growth advantage on

the cell, but additional mutations (second and third ‘hits’) will likely be required for a

tumor to develop. Rarely, retroviruses can promote transformation by inactivating a

tumor suppressor gene, though in this case the other allele must be inactivated as

well.

In a number of clinical trials of retroviral vectors, insertional activation has

resulted in adverse events. In the SCID-X1 trial, for example, 5 of 19 children treated

with a gammaretroviral vector containing the common cytokine receptor γc chain

went on to develop leukemia [163-165]. Analysis of the genomic sites of vector

integration can provide evidence of insertional activation and shed light on the

mechanism of oncogenesis. Integration events that alter the expression of cellular

harboring them. Those cells would therefore accumulate in the treated individual,

and be more frequently recovered upon random sampling of circulating cells and

bone marrow. Such analysis has been carried out for a number of gene therapy trials.

In the SCID-X1 trial, for example, samples from patients who developed leukemia

exhibited integration sites within or near the known growth-control genes LMO2,

BMI1 and CCND2 in blast cells [164]. Clonal dominance was also observed in a trial

of a gammaretroviral vector administered for the treatment of chronic granulomatous

disease, which progressed to leukemia. In this case, both patients developed a clonal

expansion of myeloid cells bearing integration sites in MDS1/EVI1, PDRM16 or

SETBP1 and myelodysplasia [166, 167].

The factors determining the incidence and consequences of insertional

activation are not fully understood, but are likely a combination of vector regulatory

elements, the nature of the transgene, the culture and transduction conditions

employed and characteristics of the target cell [168].

The contribution of cell-intrinsic factors to the incidence of insertional

activation is relatively poorly characterized. The self-renewal properties of the target

cell likely affect the consequences of vector integration. For example, Recchia and

colleagues reported [169] that gammaretroviral transduction of terminally

differentiated T-cells, though altering the expression of a large number of cellular

genes, did not result in clonal skewing as long as 9 years post-transplantation.

Similarly, Kustikova et al. reported that clonal dominance developed following

lineage-restricted progenitors [170].

Likewise, the nature of the transgene and the nature of the disorder being treated is thought to affect the potential for insertional activation. For example, in the SCID-X1 trial, it is likely that the fact the γc chain was required for the survival of the

targeted cells and that corrected cells expanded to fill an empty hematological compartment meant that transduced cells already had a growth advantage, increasing the selective forces driving clonal outgrowth [168].

The determinant of insertional activation viewed as the most straightforward to control is vector design. One proposed approach to reducing the risk of gene therapy would be to target integration events to specific sites in the genome, chosen to lie far from growth-control genes to minimize the risk of insertional activation. This is not yet a practical approach, but some success has been achieved creating chimeric proteins to retarget integration. Chapter 3 of this dissertation describes retargeting of lentiviral integration out of transcription units using such a LEDGF/p75 fusion.

In the absence of targeted integration, numerous vector design modifications have been proposed to reduce the vector’s impact on the expression of nearby genes. Notably, many of these features were not present in the vectors used in the clinical trials and resulting adverse events described above [166, 171]. Use of physiologic

cellular promoters such as PGK or EF1α to drive transgene expression, rather than

strong retroviral promoters, has been shown to reduce transactivation [172]. The inclusion of insulator elements in vectors, in addition to reducing transgene silencing,

reduces activation of neighboring genes by proviral promoter and enhancer elements

[173, 174]. Deletion of U3, which contains the viral promoter sequence, from the 3’

LTR of the viral genome reduces transactivation of neighboring genes [175, 176].

Studies of insertional activation with such vectors, termed ‘self-inactivating’ (SIN),

have supported the idea that they are less genotoxic [172, 177], though there remain

examples of tumor development with SIN vectors [173, 178]. Insertional activation

by leaky vector transcription can be reduced by the incorporation of exogenous

polyadenylation signals such as that of SV40 in addition to that in the 3’ U5 [179].

Finally, lentiviral vectors have been proposed to be safer than gammaretroviral

vectors, as discussed below.