Background
Upon encountering foreign invaders, dendritic cells (DCs) in the periphery of the body undergo a dynamic and coordinated reprogramming of gene expression, surface phenotype and cellular function [
1]. While this maturation is ongoing, DCs migrate to lymphoid organs where they interact with T lymphocytes which, in turn, decode the DC message to start a cascade of events ultimately leading to immune responses against the invading antigens. Thus, at least theoretically, safe and effective systems for delivering antigenic and/or adjuvant proteins/genes to DCs, T cells or both represent valuable means of eliciting and modulating type, extent, and duration of adaptive immune responses [
2]. Although initial attempts to achieve this goal using conventional methods were disappointing, recent advances have opened new and more promising avenues [
3,
4].
Viruses are considered ideal for delivering transgenes due to their inherent ability to bring genetic material into cells but need extensive engineering to overcome limitations such as the spectrum of cells they can enter and the noxious effects they may exert. For example, adenoviral vectors have been shown to be effective at transducing DCs and T lymphocytes [
5] but, on a negative side, they have been seen to induce massive production of proinflammatory cytokines and robust vector-specific immune responses [
6]. On the other hand, oncoretroviral vectors interfere minimally with normal body and cell functions but are poor at transducing nondividing and rarely dividing cells, such as DCs and resting T cells.
Consistent with the ability of lentiviral genomes to reach the nucleus of host cells even if these do not divide [
7], vectors derived from the human immunodeficiency virus (HIV) have been found to transduce DCs and T cells at high efficiency [
8‐
11]. In their current versions, HIV vectors have most of the original viral genome deleted, including some transcriptional elements in the U3 region of the 3' long terminal repeat (LTR) of the DNA used to produce the vector RNA. During reverse transcription, this deletion is transferred to the 5' LTR of the proviral DNA, thus generating two LTRs which are mostly inactive (self-inactivating [SIN] vectors). Also, these vectors are produced using multiple constructs encoding different components to minimize the risk of generating replication-competent viruses.
Because of safety concerns [
12], vectors derived from the feline immunodeficiency virus (FIV) are considered a good alternative to the HIV vectors because FIV has never been detected in animal species other than domestic and wild cats and has similar genome organization but minimal sequence homology to HIV, thus minimizing the risk of unwanted recombinations [
13].
The FIV vectors described to date have been very successful at delivering transgenes into a variety of cells of different animal species [reviewed in [
12]] but have performed poorly when used to transduce DCs, T lymphocytes and non-adherent white blood cells in general [
14‐
16]. In this report, we describe a SIN FIV vector that effectively transduces
ex vivo murine DCs and T cells.
Discussion
Lentivirus-derived vectors possess several advantages, including that they ensure stable and tightly controlled expression of transgenes by integrating into the cell genome, integrate preferentially into actively transcribed genes yet distantly from cellular promoters [
35‐
37], transduce quiescent and dividing cells alike [
7,
30,
38], and possibly have a lower insertional mutagenesis risk relative to vectors derived from other retroviruses [
39]. Among such vectors, those derived from FIV have been shown to be as efficient as HIV vectors at transducing a variety of cell types and tissue compartments
in vivo and have the added advantage of posing less safety concerns [
12,
28]. However, the FIV vectors described to date performed poorly when used for transducing immune cells [
14‐
16,
40], a limitation that prompted us to develop an FIV vector that might efficiently and stably deliver genes into DCs and T cells.
The vector we first constructed, LA34 is entirely derived from an FIV strain known to be much attenuated compared to field isolates [
17,
30] and, to further increase its safety, is self-inactivated by bearing LTRs partially deleted and totally inactive. The expression construct could be easily pseudotyped with two distinct Envs that conferred either a broad or a more restricted cell tropism. With the aim to obtain a vector that could be used in mouse models, efficiency at transducing the murine cell line NIH-3T3 was a major guiding criterion in its design as well as in optimizing transduction protocol. However, in its original format LA34 performed poorly with NIH-3T3 cells. Thus, in the attempt to overcome this drawback, the following modifications were introduced:
1) lentiviruses have evolved a PIC consisting of cellular and viral proteins which effectively delivers viral cDNA in close proximity of the cell genome. Since LA34 lacked cPPT, one of the two single-strand flaps generated during reverse transcription that are thought to optimize cDNA folding and enhance its steric fit in the nuclear pore [
29], we inserted it between RRE and expression cassette. In contrast to what observed with other FIV vector formats [
28,
30], this modification failed to improve LA34 performances. The reason(s) was not addressed, but it is plausible that p34TF10, the parental clone from which LA34 was produced, is
per se minimally dependent on this motif or that the p34TF10-derived cPPT we used, being slightly different in sequence from the ones previously used [
30], was poorly functional.
2) the genome of lentiviruses is encapsidated through the mutual recognition of specific RNA sequences (
ψ) and specific viral and cellular proteins [
41]. In FIV,
ψ is believed to consist of at least two discontinuous core regions, the first located upstream the major splice donor site, the second extending into
gag. While later analyses have located the principal
ψ domains within the first 100–120 nt [
31], in early RNA protection experiments, optimal viral RNA encapsidation was obtained by extending the
gag region to 311 nt [
23]. A 310-nt sequence, that in our FIV encompasses the entire region found to be important in the above study [
23], was then substituted for the 120-nt sequence present in LA34, but this brought no benefits and actually impaired nonfeline cell transduction. Again, the reason(s) for this discrepancy relative to previous studies was not investigated but it might lie in differences between the FIV clones used or in the genomic organization of the vectors developed. In any case, these results, together with the reported presence of negative regulatory sequences upstream and within
ψ [
31,
42] and the consideration that fewer parental virus sequences are present in a vector the better, made us to decide in favor of the vector with the shorter
ψ.
3) the introduction of the RNA transport element WPRE into FIV- and HIV-derived vectors has been shown to stabilize and enhance transgene expression [
30,
43]. When we introduced this element into LA34 we also observed a favorable impact on vector performance: the vector thus modified (LAW34) not only showed higher titers relative to LA34 but also transduced with enhanced efficiency all the cell lines. Importantly, the positive effects of WPRE were most evident in NIH-3T3 cells, suggesting that LAW34 might be particularly suitable for transducing murine cells.
We then focused on further optimizing LAW34 for usage with murine NIH-3T3 cells by modifying external coat and transduction protocol, two factors known to be as important as the vector itself for successful gene targeting and delivery. VSV-G pseudotyping is extremely convenient for vector development and
in vitro testing since, being the cell receptor for VSV a ubiquitous phospholipid [
44], it allows vector access into virtually any cell type, but this extended host range can be unpractical for vector usage
in vivo where restricted cell targeting is often required. Moreover, VSV-G pseudotyped vectors have proved toxic for
ex vivo cells and prone to inactivation by complement [
45]. Thus, while in setting up the vector we had used VSV-G pseudotyped particles, in subsequent studies, among the many Env glycoproteins available for pseudotyping retrovirus vectors particles [
46], we selected RD114/TR, derived from the feline endogenous ecotropic virus RD114, which has a sodium-dependent neutral-amino-acid transporter as cellular receptor, but having the cytoplasmatic tail of MLV-A. This chimeric Env, which in other systems has been shown to substantially increase vector titers, improve cellular localization and interaction with Gag [
47], resist lysis by human complement and permit transduction of primary human lymphocytes and stem cells [
24], to our knowledge had never been used with FIV vectors. The choice proved fortunate, because LAW34 pseudotyped with this Env (LAW34/RD114/TR) transduced at high efficiency all the cell lines tested and compared favorably to VSV-G pseudotyping regardless of the transducing protocol used.
We finally examined the efficiency of LAW34 at transducing
ex vivo murine DCs and T cells using different transduction protocols. The results showed that the vector has the ability to efficiently transduce both cell types, but that the Env with which it is pseudotyped is critical for DC transduction. Indeed, while T cells were transduced to similar extents regardless of whether the vector was coated with VSV-G or RD114/TR, DCs were transduced by LAW34/RD114/TR alone, thus suggesting that previous unsatisfactorily attempts to efficiently transduce DCs with FIV vectors [
14‐
16,
40] may have been due to the use of Envs that did not permit proper entry into these cells. Of note, in agreement with findings showing that DCs transduced with different vectors develop a mature phenotype [
1,
6,
11], murine DCs transduced with LAW34/RD114/TR exhibited at least some of the morphological and surface markers known to accompany DC maturation, thus showing that they were still functional. A further observation of interest was that LAW34 transduction of DCs and T cells occurred also in the absence of added facilitating agents, such as PB, that preferably should be avoided when vectors are used
in vivo.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
All Authors read and approved the final manuscript. MP is the project leader and wrote the paper, LV and AR constructed the vector and made substantial contribution to its design, FB designed and constructed the packaging vectors, FC and BDS were involved in the in vitro characterization of the vector, GF produced and analyzed the dendritic cells, MB was involved in drafting and critical revising the manuscript.