**Gene Therapy of Hematopoietic and Immune Systems: Current State and Perspectives**

Maria Savvateeva1, Fedor Rozov1,2 and Alexander Belyavsky1 *1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences 2University of Oslo, Centre for Medical Studies Russia, Moscow Russian Federation* 

#### **1. Introduction**

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Hematopoietic stem cells (HSCs) present arguably the best entry point for gene therapy of hematopoietic and immune systems since genetically modified HSCs are long-lived and would eventually transfer the therapeutic constructs to all their descendants. However, gene therapy via HSCs, although conceptually simple, has proven to be a technically formidable problem that has yet to be solved successfully. Despite overtly positive results obtained in gene therapy experiments performed with mouse and larger animal models, these achievements did not translate into clinically acceptable outcomes for non-human primates and human patients, with exception of a few specific disease instances where a therapeutic gene brought about significant survival advantages to transduced cells (Cavazzana-Calvo et al., 2000, Schmidt et al, 2003). Major differences between outcomes of conceptually similar experiments in mice and primates underscore the notion that the fundamental principles governing functioning of hematopoietic system in small short-lived vs. larger long-lived animals differ significantly. Low degree of chimerism obtained in experiments with primates and humans is likely a result of intrinsically low efficiency of viral transduction of long-term repopulating (LTR) HSCs coupled with subsequent massive silencing of integrated constructs (Ellis, 2005; Horn et al, 2002). One may hypothesize that this situation reflects a better protection of hematopoietic system from external influences, in particular invasion of foreign genetic material, in longer-living animals.

However, our deepening knowledge of molecular mechanisms underlying functioning of HSCs within the organism provides hints as to what strategies may lead to the development of the efficient gene therapy via HSCs; some of these strategies are discussed below.

#### **2. Improvements of vectors and ex vivo HSC transduction protocols**

Numerous studies indicate that lentiviral vectors that are capable of transducing nondividing cells may represent a more promising tool for introduction of genetic material into HSCs compared to retroviral vectors (Uchida et al, 1998, Case et al., 1999). This may be attributed to a largely quiescent nature of LTR HSCs, especially in larger animals (Cheshier et al., 1999, Shepherd et al., 2007). Since even lentiviral vectors transduce more efficiently dividing cells than quiescent ones (Trobridge et al., 2004), the current transduction protocols relied until recently on the use of culture conditions that induced entry of HSCs into cell

Gene Therapy of Hematopoietic and Immune Systems: Current State and Perspectives 443

marker Selecting agent Mode of action References

BCNU or TMZ

(5-FU) and related

DHFR. Drug resistant

efflux pump

Some other members of the HOX family, either alone or fused with specific cellular partners, are also able to induce expansion of hematopoietic progenitors in mice. Of particular importance is a fusion gene NUP98-HoxA10, which has a remarkable ability of multi-log expansion of murine repopulating cells ex vivo, exceeding that of HoxB4 (Ohta et al., 2007;

Recently, the powerful effect of overexpression of early acting transcription factor SALL4 on ex vivo expansion of human hematopoietic cells capable of long-term repopulation of NOD/SCID mice was demonstrated (Aguila et al., 2011). Significant ex vivo expansion

There are at least a dozen of other genes that, when overexpressed, induce significant expansion of HSCs in mice in vivo. One of the most interesting groups of such factors are epigenetic regulators. Of particular interest is Bmi1, a member of Polycomb group, which is involved in regulation of mantenance of various adult stem cell types. Inactivation of Bmi1

Table 1. Strategies for negative selection of genetically modified HSC

could be also achieved using recombinant TAT-SALL4B protein.

death.

MGMT protein functions to repair

Drug-resistant TS can protect bone marrow cells from 5-fluorouracil

fluoropyrimidines that induce cessation of DNA and RNA synthesis, and subsequent cell

MTX acts on highly proliferative cells, blocking DNA synthesis through competitive inhibition of

dihydrofolate reductase such as Tyr22 (Tyr22DHFR) has the potential to selectively increase engraftment of gene-modified human hematopoietic cells

Overexpression of the multidrug resistance gene MDR1 in bone marrow cells results in protection from hematopoietic toxicity from chemotherapy drugs that are substrates for the MDR1 drug

Sawai et al, 2001; Zielske et

Bielas et al, 2009

Gori et al, 2010

Cowan et al,

1999

al, 2003

alkylated DNA caused by chemotherapeutic agents like

Slective

BCNU, TMZ, other alkylating

5-fluorouracil (5-FU)

5-fluorodeoxyuridine (5- FUdR)

Taxol, Paclitaxel

agents

Tyr22DHFR Methotrexate

O6-MGMT

Thymidylate synthase

Multidrug resistance gene-1 (MDR)

Watts et al., 2011).

cycle but incidentally failed to maintain their stem cell status (Bunting et al., 1999). This situation seems to have been ameliorated after introduction of transduction protocols that rely on the use of serum-free media that lack factors inducing SC differentiation (Mostoslavsky et al., 2005) and novel growth factors that better preserve cell stemness (Zhang C et al., 2008). It remains yet to see whether these improvements are sufficient to significantly increase the efficiency of HSC gene therapy in clinical settings.
