Optimizing lentiviral vector formulation conditions for efficient ex vivo transduction of primary human T cells in chimeric antigen receptor T-cell manufacturing.

BACKGROUND AIMS: Chimeric antigen receptor (CAR) T-cell products are commonly generated using lentiviral vector (LV) transduction. Optimal final formulation buffer (FFB) supporting LV stability during cryostorage is crucial for cost-effective manufacturing. METHODS: To identify the ideal LV FFB composition for ex vivo CAR-T production, primary human T cells were transduced with vesicular stomatitis virus G-protein (VSV-G) -pseudotyped LVs (encoding a reporter gene or an anti-CD19-CAR). The formulations included phosphate-buffered saline (PBS), HEPES, or X-VIVOTM 15, and stabilizing excipients. The functional and viral particle titers and vector copy number were measured after LV cryopreservation and up to 24 h post-thaw incubation. CAR-Ts were produced with LVs in selected FFBs, and the resulting cells were characterized. RESULTS: Post-cryopreservation, HEPES-based FFBs provided higher LV functional titers than PBS and X-VIVOTM 15, and 10% trehalose-20 mM MgCl2 improved LV transduction efficiency in PBS and 50 mM HEPES. Thawed vectors remained stable at +4°C, while a ≤ 25% median decrease in the functional titer occurred during 24 h at room temperature. Tested excipients did not enhance LV post-thaw stability. CAR-Ts produced using LVs cryopreserved in 10% trehalose- or sucrose-20 mM MgCl2 in 50 mM HEPES showed comparable transduction rates, cell yield, viability, phenotype, and in vitro functionality. CONCLUSION: A buffer consisting of 10% trehalose-20 mM MgCl2 in 50 mM HEPES provided a feasible FFB to cryopreserve a VSV-G -pseudotyped LV for CAR-T-cell production. The LVs remained relatively stable for at least 24 h post-thaw, even at ambient temperatures. This study provides insights into process development, showing LV formulation data generated using the relevant target cell type for CAR-T-cell manufacturing.

Corrigendum: Optimized riboswitch-regulated AAV vector for VEGF-B gene therapy.

[This corrects the article DOI: 10.3389/fmed.2022.1052318.].

Optimized riboswitch-regulated AAV vector for VEGF-B gene therapy.

Gene therapy would greatly benefit from a method to regulate therapeutic gene expression temporally. Riboswitches are small RNA elements that have been studied for their potential use in turning transgene expression on or off by ligand binding. We compared several tetracycline and toyocamycin-inducible ON-riboswitches for a drug responsive transgene expression. The tetracycline-dependent K19 riboswitch showed the best control and we successfully applied it to different transgenes. The induction of gene expression was 6- to 10-fold, dose-dependent, reversible, and occurred within hours after the addition of a clinically relevant tetracycline dose, using either plasmid or adeno-associated virus (AAV) vectors. To enhance the switching capacity, we further optimized the gene cassette to control the expression of a potential therapeutic gene for cardiovascular diseases, VEGF-B. Using two or three riboswitches simultaneously reduced leakiness and improved the dynamic range, and a linker sequence between the riboswitches improved their functionality. The riboswitch function was promoter-independent, but a post-transcriptional WPRE element in the expression cassette reduced its functionality. The optimized construct was a dual riboswitch at the 3' end of the transgene with a 100 bp linker sequence. Our study reveals significant differences in the function of riboswitches and provides important aspects on optimizing expression cassette designs. The findings will benefit further research and development of riboswitches.

Functional roles of the membrane-associated AAV protein MAAP.

With a limited coding capacity of 4.7 kb, adeno-associated virus (AAV) genome has evolved over-lapping genes to maximise the usage of its genome. An example is the recently found ORF in the cap gene, encoding membrane-associated accessory protein (MAAP), located in the same genomic region as the VP1/2 unique domain, but in a different reading frame. This 13 KDa protein, unique to the dependovirus genus, is not homologous to any known protein. Our studies confirm that MAAP translation initiates from the first CTG codon found in the VP1 ORF2. We have further observed MAAP localised in the plasma membrane, in the membranous structures in close proximity to the nucleus and to the nuclear envelope by co-transfecting with plasmids encoding the wild-type AAV (wt-AAV) genome and adenovirus (Ad) helper genes. While keeping VP1/2 protein sequence identical, both inactivation and truncation of MAAP translation affected the emergence and intracellular distribution of the AAV capsid proteins. We have demonstrated that MAAP facilitates AAV replication and has a role in controlling Ad infection. Additionally, we were able to improve virus production and capsid integrity through a C-terminal truncation of MAAP while other modifications led to increased packaging of contaminating, non-viral DNA. Our results show that MAAP plays a significant role in AAV infection, with profound implications for the production of therapeutic AAV vectors.

Experimental Evaluation of an Interferometric Light Microscopy Particle Counter for Titering and Characterization of Virus Preparations.

Virus particle concentration is a critical piece of information for virology, viral vaccines and gene therapy research. We tested a novel nanoparticle counting device, "Videodrop", for its efficacy in titering and characterization of virus particles. The Videodrop nanoparticle counter is based on interferometric light microscopy (ILM). The method allows the detection of particles under the diffraction limit capabilities of conventional light microscopy. We analyzed lenti-, adeno-, and baculovirus samples in different concentrations and compared the readings against traditional titering and characterization methods. The tested Videodrop particle counter is especially useful when measuring high-concentration purified virus preparations. Certain non-purified sample types or small viruses may be impossible to characterize or may require the use of standard curve or background subtraction methods, which increases the duration of the analysis. Together, our testing shows that Videodrop is a reasonable option for virus particle counting in situations where a moderate number of samples need to be analyzed quickly.

Evaluation of the Single-Use Fixed-Bed Bioreactors in Scalable Virus Production.

The accelerating development of gene therapy from research towards clinical trials and beyond has elevated the demand for practical viral vector-manufacturing solutions. The use of disposable upstream technology is gaining traction in clinical manufacturing. Packed-bed or fixed-bed reactors, where column is packed with immobilized biocatalyst particles providing surface to constrain the cells in a particular region of the reactor, have been widely used in bioprocessing applications since mid-1900s. However, the world's first single-use, fully integrated, high cell density, fixed-bed bioreactor was launched only approximately a decade ago. By now, several single-use, fixed-bed technology solutions have been developed in a small scale. Scaling-up the manufacturing can be challenging and for commercial-scale manufacturing, there is practically only one single-use, good manufacturing practice-compliant option available. This study reviews the latest, fully disposable, fixed-bed bioreactors; compares the virus production in the different systems; and discusses important manufacturing cost-related topics. It is predicted that single-use, fixed-bed bioreactors will receive even more attention in the field of viral vector manufacturing and commercialization, especially with the need for higher virus titers and virus yields.

Development of Large-Scale Downstream Processing for Lentiviral Vectors.

The interest in lentiviral vectors (LVs) has increased prominently for gene therapy applications, but few have reached the later stages of clinical trials. The main challenge has remained in scaling up the manufacturing process for the fragile vector to obtain high titers for in vivo usage. We have previously scaled up the LV production to iCELLis 500, being able to produce up to 180 L of harvest material in one run with perfusion. The following challenge considers the purification and concentration of the product to meet titer and purity requirements for clinical use. We have developed a downstream process, beginning with clarification, buffer exchange, and concentration, by tangential flow filtration. This is followed by a purification step using single membrane-based anion exchange chromatography and final formulation with tangential flow filtration. Different materials and conditions were compared to optimize the process, especially for the chromatography step that has been the bottleneck in lentiviral vector purification scale-up. The final infectious titer of the lentiviral vector product manufactured using the optimized scale-up process was determined to be 1.97 × 109 transducing units (TU)/mL, which can be considered as a high titer for lentiviral vectors.

Benchmarking of Scale-X Bioreactor System in Lentiviral and Adenoviral Vector Production.

We have previously produced viral vectors (lentiviral vector, adenoviral vector, and adeno-associated viral vector) in small and in commercial scale in adherent cells using Pall fixed-bed iCELLis® bioreactor. Recently, a company called Univercells has launched a new fixed-bed bioreactor with the same cell growth surface matrix material, but with different fixed-bed structure than is used in iCELLis bioreactor. We sought to compare the new scale-X™ hydro bioreactor (2.4 m2) and iCELLis Nano system (2.67 m2) to see if the difference has any effect on cell growth or lentiviral vector and adenoviral vector productivity. Runs were performed using parameters optimized for viral vector production in iCELLis Nano bioreactor. Cell growth was monitored by counting nuclei, as well as by following glucose consumption and lactate production. In both bioreactor systems, cells grew well, and the cell distribution was found quite homogeneous in scale-X bioreactor. Univercells scale-X bioreactor was proven to be at least equally efficient or even improved in both lentiviral vector and adenoviral vector production. Based on the results, the same protocol and parameters used in viral vector production in iCELLis bioreactor can also be successfully used for the production in scale-X bioreactor system.

Detection of lentiviral suicide gene therapy in C6 rat glioma using hyperpolarised [1-13 C]pyruvate.

Hyperpolarised [1-13 C]pyruvate MRI has shown promise in monitoring therapeutic efficacy in a number of cancers including glioma. In this study, we assessed the pyruvate response to the lentiviral suicide gene therapy of herpes simplex virus-1 thymidine kinase with the prodrug ganciclovir (HSV-TK/GCV) in C6 rat glioma and compared it with traditional MR therapy markers. Female Wistar rats were inoculated with 106 C6 glioma cells. Treated animals received intratumoural lentiviral HSV-TK gene transfers on days 7 and 8 followed by 2-week GCV therapy starting on day 10. Animals were repeatedly imaged during therapy using volumetric MRI, diffusion and relaxation mapping, as well as metabolic [1-13 C]pyruvate MRS imaging. Survival (measured as time before animals reached a humane endpoint and were euthanised) was assessed up to day 30 posttherapy. HSV-TK/GCV gene therapy lengthened the median survival time from 12 to 25 days. This was accompanied by an apparent tumour growth arrest, but no changes in diffusion or relaxation parameters in treated animals. The metabolic response was more evident in the case-by-case analysis than in the group-level analysis. Treated animals also showed a 37 ± 15% decrease (P < 0.05, n = 5) in lactate-to-pyruvate ratio between therapy weeks, whereas a 44 ± 18% increase (P < 0.05, n = 6) was observed in control animals. Hyperpolarised [1-13 C]pyruvate MRI can offer complementary metabolic information to traditional MR methods to give a more comprehensive picture of the slowly developing gene therapy response. This may benefit the detection of the successful therapy response in patients.

Preclinical Proof-of-Concept, Analytical Development, and Commercial Scale Production of Lentiviral Vector in Adherent Cells.

The therapeutic efficacy of a lentiviral vector (LV) expressing the herpes simplex virus thymidine kinase (HSV-TK) was studied in an immunocompetent rat glioblastoma model. Intraperitoneal ganciclovir injections (50 mg/kg/day) were administered for 14 consecutive days, resulting in reduced tumor volumes as monitored by MRI. Survival analyses revealed a significant improvement among the LV-expressing HSV-TK (LV-TK)/ganciclovir-treated animals when compared to non-treated control rats. However, a limiting factor in the use of LV has been the suboptimal small-scale production in flasks. Our aim during the translation phase, prior to entering the final pre-clinical and early clinical phases, was to develop a scalable, robust, and disposable manufacturing process for LV-TKs. We also aimed to minimize future process changes and enable production upscaling to make the process suitable for larger patient populations. The upstream process relies on fixed-bed iCELLis technology and transient plasmid transfection. This is the first time iCELLis 500 commercial-scale bioreactor was used for LV production. A testing strategy to determine the pharmacological activity of LV-TK drug product by measuring cell viability was developed, and the specificity of the potency assay was also proven. In this paper we focus on upstream process development while showing analytical development and the proof-of-concept of LV-TK functionality.

Comparative Study of Adeno-associated Virus, Adenovirus, Bacu lovirus and Lentivirus Vectors for Gene Therapy of the Eyes.

BACKGROUND: The eye possesses unique anatomical features that make it a valuable target for gene therapy applications. OBJECTIVE: The aim of the current study was to compare transduction efficiency, safety and biodistribution of four viral vectors following intravitreal injection. METHOD: Adenovirus (AdV), Adeno-Associated Virus (AAV), Baculovirus (BV) and Lentivirus (LV) vectors encoding Green Fluorescent Protein (GFP) were injected bilaterally intravitreally into adult C57BL/6OlaHsd mice. Control mice received saline. Eyes and other organs were studied at multiple time points from 3 days to 6 months. Immunohistochemical stainings with retinal cell markers were performed to verify GFP-positive cells. Biodistribution in retina and various non-target tissues was studied using a qPCR method. Inflammatory responses and toxicity were investigated from cryostat eye sections and serum samples. RESULTS: AAV-injected eyes showed GFP expression both in inner and outer retinal cells from 7 days up to 6 months. LV eyes showed long lasting transgene expression mostly in retinal pigment epithelium whereas AdV transiently transduced mainly cells in the anterior chamber. In BV-injected eyes, GFP positivity was very low. qPCR results showed that AdV, AAV and LV spread into the optic nerve, but were below the detection limit in other organs. The strongest immune responses were evoked by intravitreal injections of AdV and BV. The highest concentration of anti-GFP IgG was detected in the AdV-treated group, whereas the AAV group showed the lowest concentration. Neither blood chemistry screen nor the number of apoptotic cells showed any differences between the viral vector and saline injected groups. CONCLUSION: Our findings show that intravitreal gene delivery is safe and feasible with AAV, AdV and lentivirus vectors.

Tissue damage drives co-localization of NF-κB, Smad3, and Nrf2 to direct Rev-erb sensitive wound repair in mouse macrophages.

Although macrophages can be polarized to distinct phenotypes in vitro with individual ligands, in vivo they encounter multiple signals that control their varied functions in homeostasis, immunity, and disease. Here, we identify roles of Rev-erb nuclear receptors in regulating responses of mouse macrophages to complex tissue damage signals and wound repair. Rather than reinforcing a specific program of macrophage polarization, Rev-erbs repress subsets of genes that are activated by TLR ligands, IL4, TGFß, and damage-associated molecular patterns (DAMPS). Unexpectedly, a complex damage signal promotes co-localization of NF-κB, Smad3, and Nrf2 at Rev-erb-sensitive enhancers and drives expression of genes characteristic of multiple polarization states in the same cells. Rev-erb-sensitive enhancers thereby integrate multiple damage-activated signaling pathways to promote a wound repair phenotype.

Process Development of Adenoviral Vector Production in Fixed Bed Bioreactor: From Bench to Commercial Scale.

Large-scale vector manufacturing for phase III and beyond has proven to be challenging. Upscaling the process with suspension cells is increasingly feasible, but many viral production applications are still applicable only in adherent settings. Scaling up the adherent system has proven to be troublesome. The iCELLis(®) disposable fixed-bed bioreactors offer a possible option for viral vector manufacturing in large quantities in an adherent environment. In this study, we have optimized adenovirus serotype 5 manufacturing using iCELLis Nano with a cultivation area up to 4 m(2). HEK293 cell cultivation, infection, and harvest of the virus (by lysing the cells inside the bioreactor) proved possible, reaching total yield of up to 1.6×10(14) viral particles (vp)/batch. The iCELLis 500 is designed to satisfy demand for large-scale requirements. Inoculating a large quantity of cell mass into the iCELLis 500 was achieved by first expanding the cell mass in suspension. Upscaling the process into an iCELLis 500/100 m(2) cultivation area cassette was practical and produced up to 6.1×10(15) vp. Flask productivity per cm(2) in iCELLis Nano and iCELLis 500 was in the same range. As a conclusion, we showed for the first time that iCELLis 500 equipment has provided an effective way to manufacture large batches of adenoviral vectors.

Differential Promoter Methylation of Macrophage Genes Is Associated With Impaired Vascular Growth in Ischemic Muscles of Hyperlipidemic and Type 2 Diabetic Mice: Genome-Wide Promoter Methylation Study.

RATIONALE: Hyperlipidemia and type 2 diabetes mellitus (T2DM) severely impair adaptive vascular growth responses in ischemic muscles. This is largely attributed to dysregulated gene expression, although details of the changes are unknown. OBJECTIVE: To define the role of promoter methylation in adaptive vascular growth in hyperlipidemia (LDLR(-/-)ApoB(100/100)) and T2DM (IGF-II/LDLR(-/-)ApoB(100/100)) mouse models of hindlimb ischemia. METHODS AND RESULTS: Unilateral hindlimb ischemia was induced by ligating femoral artery. Perfusion was assessed using ultrasound, and capillary and arteriole parameters were assessed using immunohistochemistry. Genome-wide methylated DNA sequencing was performed with DNA isolated from ischemic muscle, tissue macrophages (Mϕs), and endothelial cells. Compared with the controls, hyperlipidemia and T2DM mice showed impaired perfusion recovery, which was associated with impaired angiogenesis and arteriogenesis. Genome-wide proximal promoter DNA methylation analysis suggested differential patterns of methylation in Mϕ genes in ischemic muscles. Classically activated M1-Mϕ gene promoters, including Cfb, Serping1, and Tnfsf15, were significantly hypomethylated, whereas alternatively activated M2-Mϕ gene promoters, including Nrp1, Cxcr4, Plxnd1, Arg1, Cdk18, and Fes, were significantly hypermethylated in Mϕs isolated from hyperlipidemia and T2DM ischemic muscles compared with controls. These results combined with mRNA expression and immunohistochemistry showed the predominance of proinflammatory M1-Mϕs, compared with anti-inflammatory and proangiogenic M2-Mϕs in hyperlipidemia and T2DM ischemic muscles. CONCLUSIONS: We found significant promoter hypomethylation of genes typical for proinflammatory M1-Mϕs and hypermethylation of anti-inflammatory, proangiogenic M2-Mϕ genes in hyperlipidemia and T2DM ischemic muscles. Epigenetic alterations modify Mϕ phenotype toward proinflammatory M1 as opposed to anti-inflammatory, proangiogenic, and tissue repair M2 phenotype, which may contribute to the impaired adaptive vascular growth under these pathological conditions.

Soluble vascular endothelial growth factor receptor-1 improves therapeutic efficacy of suicide gene therapy in an angiogenesis-independent manner.

Abstract Malignant gliomas (MGs) are highly vascularized, aggressive brain cancers carrying a dismal prognosis. Because of their high vascularity, anti-angiogenic therapy is a potential treatment option. Indeed, the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab has demonstrated promising results in clinical trials. Similarly, adenovirus-medicated Herpes simplex virus thymidine kinase and ganciclovir (AdHSV-tk/GCV) suicide gene therapy has established itself in clinical trials as a potential novel therapeutic strategy for MGs. In this study, we demonstrate the feasibility of combining adenovirus-mediated soluble VEGF receptor-1 anti-angiogenic gene therapy with AdHSV-tk/GCV suicide gene therapy to treat experimental MGs. Our results reveal that, apart from inhibiting angiogenesis, other anti-tumor mechanisms, such as reduction of infiltration by tumor-associated macrophages/microglia, may contribute to the improved therapeutic benefit of combination therapy.

Lentiviral protein transduction with genome-modifying HIV-1 integrase-I-PpoI fusion proteins: studies on specificity and cytotoxicity.

Rare-cutting endonucleases, such as the I-PpoI, can be used for the induction of double strand breaks (DSBs) in genome editing and targeted integration based on homologous recombination. For therapeutic approaches, the specificity and the pattern of off-target effects are of high importance in these techniques. For its applications, the endonuclease needs to be transported into the target cell nucleus, where the mechanism of transport may affect its function. Here, we have studied the lentiviral protein transduction of the integrase (IN)-PpoI fusion protein using the cis-packaging method. In genome-wide interaction studies, IN-fusion proteins were verified to bind their target sequence containing 28S ribosomal RNA (rRNA) genes with a 100-fold enrichment, despite the well-documented behavior of IN to be tethered into various genomic areas by host-cell factors. In addition, to estimate the applicability of the method, DSB-induced cytotoxic effects with different vector endonuclease configurations were studied in a panel of cells. Varying the amount and activity of endonuclease enabled the adjustment of ratio between the induced DSBs and transported DNA. In cell studies, certain cancerous cell lines were especially prone to DSBs in rRNA genes, which led us to test the protein transduction in a tumour environment in an in vivo study. In summary, the results highlight the potential of lentiviral vectors (LVVs) for the nuclear delivery of endonucleases.

Biodistribution and antitumor effect of Cetuximab-targeted lentivirus.

Viral vectors are central tools for gene therapy. Targeting of the vector to desired tissues followed by expression of the therapeutic gene forms one of the most critical points in effective therapy. In this study we used streptavidin-displaying lentivirus conjugated to biotinylated anti-epidermal growth factor receptor (EGFR) antibody (Cetuximab) to target vector specifically to ovarian tumors. Biodistribution of the targeted virus was studied in nude mice with orthotropic SKOV-3m human ovarian carcinoma xenografts. Radiolabeled antibodies were conjugated to streptavidin-displaying lentiviruses and biodistribution of the virus after the intravenous delivery to tumor-bearing mice was monitored up to 6 days using combined SPECT/CT imaging modality. Organ samples were collected post mortem and specific organ activities were measured. The integration of lentivirus vectors in collected tissue samples was analyzed using qPCR and the expression of green fluorescent protein (GFP)-transgene was tested by enzyme-linked immunosorbent assay. Our results showed that lentiviruses conjugated to Cetuximab (Cet-LV) or control human IgG (IgG-LV) accumulated mainly to the liver and spleen of the mice and to lower extent to lung, kidneys and tumors. Strikingly, in 50% of the mice injected with cetuximab-targeted lentivirus no tumor tissue was found, whereas the remaining half showed a significant decrease in tumor size. We hypothesize/present data that lentivirus-mediated INF-αß production together with tumor targeting could function as an effective antitumor treatment.

Rev-Erbs repress macrophage gene expression by inhibiting enhancer-directed transcription.

Rev-Erb-α and Rev-Erb-ß are nuclear receptors that regulate the expression of genes involved in the control of circadian rhythm, metabolism and inflammatory responses. Rev-Erbs function as transcriptional repressors by recruiting nuclear receptor co-repressor (NCoR)-HDAC3 complexes to Rev-Erb response elements in enhancers and promoters of target genes, but the molecular basis for cell-specific programs of repression is not known. Here we present evidence that in mouse macrophages Rev-Erbs regulate target gene expression by inhibiting the functions of distal enhancers that are selected by macrophage-lineage-determining factors, thereby establishing a macrophage-specific program of repression. Remarkably, the repressive functions of Rev-Erbs are associated with their ability to inhibit the transcription of enhancer-derived RNAs (eRNAs). Furthermore, targeted degradation of eRNAs at two enhancers subject to negative regulation by Rev-Erbs resulted in reduced expression of nearby messenger RNAs, suggesting a direct role of these eRNAs in enhancer function. By precisely defining eRNA start sites using a modified form of global run-on sequencing that quantifies nascent 5' ends, we show that transfer of full enhancer activity to a target promoter requires both the sequences mediating transcription-factor binding and the specific sequences encoding the eRNA transcript. These studies provide evidence for a direct role of eRNAs in contributing to enhancer functions and suggest that Rev-Erbs act to suppress gene expression at a distance by repressing eRNA transcription.

Oxidative stress-regulated lentiviral TK/GCV gene therapy for lung cancer treatment.

Nuclear factor erythroid-2 related factor 2 (Nrf2) is a transcription factor that regulates protection against a wide variety of toxic insults to cells, including cytotoxic cancer chemotherapeutic drugs. Many lung cancer cells harbor a mutation in either Nrf2 or its inhibitor Keap1 resulting in permanent activation of Nrf2 and chemoresistance. In this study, we sought to examine whether this attribute could be exploited in cancer suicide gene therapy by using a lentiviral (LV) vector expressing herpes simplex virus thymidine kinase (HSV-TK/GCV) under the regulation of antioxidant response element (ARE), a cis-acting enhancer sequence that binds Nrf2. In human lung adenocarcinoma cells in which Nrf2 is constitutively overexpressed, ARE activity was found to be high under basal conditions. In this setting, ARE-HSV-TK was more effective than a vector in which HSV-TK expression was driven by a constitutively active promoter. In a mouse xenograft model of lung cancer, suicide gene therapy with LV-ARE-TK/GCV was effective compared with LV-PGK-TK/GCV in reducing tumor size. We conclude that ARE-regulated HSV-TK/GCV therapy offers a promising approach for suicide cancer gene therapy in cells with high constitutive ARE activity, permitting a greater degree of therapeutic targeting to those cells.

Targeted delivery via avidin fusion protein: intracellular fate of biotinylated doxorubicin derivative and cellular uptake kinetics and biodistribution of biotinylated liposomes.

In this study, avidin-biotin technology was combined with a multifunctional drug carrier modality i.e. liposomes to achieve an active and versatile targeting approach. The anti-cancer drug doxorubicin (DOX) was modified with direct biotinylation (B-DOX) (Allart et al., 2003), or encapsulated in biotinylated sterically stabilized pH-sensitive liposomes (BL-DOX), and targeted to the lentiviral vector transduced cells expressing an avidin fusion protein on the cell membrane (Lehtolainen et al., 2003; Lesch et al., 2009). The direct biotinylation of doxorubicin improved cell internalization in rat glioma (BT4C) cells expressing avidin fusion protein receptor but cell toxicity was reduced by 78-fold due to impaired nuclear localization. In contrast, liposomal formulations restored the biological activity of the DOX in several cell lines. However, mainly due to uptake via non-specific pathways the active targeting of BL-DOX was negligible in both in vitro and in vivo studies. Active targeting with multifunctional drug carrier systems is challenging and further studies will be needed to optimize the properties of targeted drug carrier and receptor expression systems.