Materials promoting viral gene delivery

Therapeutic viral gene delivery is an emerging technology which aims to correct genetic mutations by introducing new genetic information to cells either to correct a faulty gene or to initiate cell death in oncolytic treatments. In recent years, significant scientific progress has led to several clinical trials resulting in the approval of gene therapies for human treatment. However, successful therapies remain limited due to a number of challenges such as inefficient cell uptake, low transduction efficiency (TE), limited tropism, liver toxicity and immune response. To adress these issues and increase the number of available therapies, additives from a broad range of materials like polymers, peptides, lipids, nanoparticles, and small molecules have been applied so far. The scope of this review is to highlight these selected delivery systems from a materials perspective.

The promise of viral gene delivery Due to their inherent infectivity, non-pathogenic viral particles (VPs) are highly interesting vectors for gene delivery. In therapeutic applications they replace or deactivate disease-causing genes or introduce new genes to treat a disease. 1 Since the first commercial production in 2003 2 the amount of gene therapies entering clinical trials and eventually earning approval is growing each year 3,4 with promising future prospects. 5 Since 2017, the European Medicine Agency (EMA) and the U.S. Food and Drug Administration (FDA) approved eight new therapeutics, 6,7 amongst them for example Luxturna®, the first adeno-associated virus-based gene therapy 8 and recently Zolgensma® a one-time injection gene therapy to treat spinal muscular atrophy. 9 Novel tailor-made gene therapeutic strategies pave the way towards personalized medicine, in which the patient's individual genetic circumstances are considered. Viral gene delivery promises to enable various new treatments and current research is directed at areas such as oncolytic virotherapy, 10 tissue regeneration and formation (bones, 11

Christopher V. Synatschke
Christopher Synatschke studied chemistry at the University of Bayreuth, Germany, and in 2013 received his PhD with distinction (summa cum laude) working on therapeutic applications of polyelectrolyte nanostructures in the group of Prof. Axel Müller. Then, as a Feodor Lynen Postdoctoral Fellow at Northwestern University, USA, he explored peptide-polymer hybrid biomaterials in the group of Prof. Samuel Stupp. After returning to Germany in 2017, he joined the group of Prof. Tanja Weil at the Max Planck Institute for Polymer Research where he now heads the "Biomaterials" group. His research interests include bioinspired, nanostructured materials to control cellular behavior. eye, 13 cardiac muscles 14 ), treatment of hemophilia, 15 neurodegenerative diseases 16 and cystic fibrosis. 17 Unfortunately, therapies in clinical studies have faced serious setbacks through side effects such as serious liver injuries. 9 A lethal case due to high dose adenovirus administration in 1999 18 raised attention to safety issues like immunogenicity, range of infectable host cells (tropism) as well to biological limitations, such as loading capacity of genetic information and batch-to-batch variation of viral vectors (VV).
Consequently, alternatives to VVs have become popular in the last 20 years. Non-viral gene delivery tries to mimic VVs by packaging genetic information in synthetic carriers like cationic polymers or lipids or by using physical methods for direct delivery to host cells as reviewed elsewhere. 19 The advantages of these approaches are a lower immunogenicity due to the lack of preformed antibodies and an easier targeted delivery owing to the modular design of synthetic carriers. 20 However, non-viral gene delivery methods often require more laborious preparation and suffer from low efficacy compared to viral methods. 21 Viruses, optimized through millions of years of evolution, still continue to be the most efficient gene carriers also for therapeutic approaches and are hence used in more than 70% of clinical trials 22 and in the majority of approved gene therapy drugs. 7

Viral vectors
Viruses are highly ordered nanoassemblies of nucleic acids and proteins and can infect a broad range of organisms from bacteria to humans, both in pathogenic and non-pathogenic contexts. 23,24 Therapeutic VVs are obtained by replacing original gene sequences with beneficial ones. 25,26 These nucleic acids are packaged in a virus capsid, which is composed of proteins displaying functional groups like amines on the surface and can interact with specific receptors on cell membranes. Some virus capsids are additionally enveloped by a lipid bilayer. 27 The virus mediated delivery of nucleic acids (transduction) consists of several steps involving diffusion and translocation through the cell membrane, followed by disruption of the endosome (endosomal escape), release and integration of viral genomes to the nucleus. 26,28 In clinical trials, adenoviruses (Ad) are the most frequently used VVs for gene transfer. Ad provides high transduction efficiency (TE) to a broad range of dividing and non-dividing cells. 29 Ad tropism is mainly determined by capsid proteins, hexones and fibers, 30,31 which contain fiber knob domains for coxsackievirus and adenovirus receptor (CAR) interaction. 32 The Ad serotype 5 (Ad5) is one of the most common vectors in clinical trials, however, they accumulate in the liver and show strong immunogenicity due to high prevalence of pre-existing immunity to Ad. 33 Adeno-associated viruses (AAV) have emerged as promising vectors with low immunogenicity and long-term stability, resulting in the recently approved therapies, marketed as Luxturna® and Zolgensma®. 34,35 As part of the retrovirus (RV) family, lentiviruses (LV) have been increasingly applied in recent years due to their inherent ability to infect both dividing and non-dividing cells, efficient integration into host genetic information and possibility for large scale production. [36][37][38][39] Finally, oncolytic viruses are attracting growing interest as versatile cancer therapeutics. [40][41][42][43][44][45] They can selectively replicate in cancer cells, eventually inducing cell-lysis and further activating antitumor immune response. 46,47 Promising oncolytic vectors are genetically modified from adenovirus, herpes virus, reovirus and measles virus. [48][49][50][51] The properties of the most common VVs discussed in this review are summarized in Table 1.

Limitations
Despite intense research efforts, VVs still face several limitations. An immune response by the host can lead to rapid inactivation of viral particles and clearance from the blood stream. Subsequent inflammatory processes can lead to liver injury or even multiorgan failure, in severe cases. 60,61 The production of neutralizing antibodies is a lasting humoral immune response, which hinders any further viral gene delivery. 62 To minimize an immune response, low vector doses are used which in turn results in reduced efficiency.
Moreover, the entry to cells can be limited by the natural tropism of VVs. For example, Ad cannot transduce to CARnegative CD34 + stem cells. 63 Further, transduction can also be hindered by low passive diffusion rates to cell membrane, 64 poor translocation to the cellular endosome, slow endosomal escape and inefficient nuclear genome integration. 65,66 Strategies to promote viral gene delivery Over several decades various techniques to promote the delivery of VVs have been developed. These can be categorized into (i) physical methods, (ii) genetic bioengineering of viruses, and (iii) chemical methods, namely material additives. The main aims of these delivery strategies include enhancing transduction efficiency (TE), long-term release of VPs, reduction of immune response, broadening of tropism and targeted delivery. 6,67 Physical methods Physical methods such as microinjection, 68,69 microfluidic, 70,71 sonication, 72,73 centrifugation, 74 cellular deformation, 75 laser irradiation 76,77 or electroporation 78,79 induce or facilitate cell entry of vectors by mechanical force, mainly through disrupting the plasma membrane. They are commonly applied in non-viral gene delivery because they transport the genetic information easily without the need for carriers and the otherwise low infectivity. These methods are also viable for viral gene delivery with electroporation being a noteworthy exception. 80 A downside of using VVs with physical methods is the lack of protection from immune response as well as the invasive and cell-damaging procedure. Very comprehensive overviews of membrane disruptive methods for cargo delivery have recently been published elsewhere. 81,82 Genetic engineering Genetic engineering of virus capsids is laborious but gives access to vectors with higher safety profile, modified tropism, and no batch-to-batch variability. Especially in recent years, highly efficient vectors could be obtained using genetic manipulation. Strategies include amino acids mutations, peptide domain insertions and incorporation of chemical functional groups [83][84][85] as recently reviewed for AAV. [86][87][88] For example, introduction of unnatural amino acid residues such as azides on the capsid surface can give access to selective click chemistry. 89,90 Further, genetically modified viruses can enhance TE, 91,92 broaden the tropism, 63,93 enable fluorescent imaging, 85 escape neutralizing antibodies, 94,95 generate stimuli responsive vectors, 96 e.g. by light 97,98 or enzymes, 99 and target cells. 100,101 Comprehensive recent reviews on bioengineering AAV can be found elsewhere. 102,103 Chemical methods Viral gene delivery can be enhanced by synthetic additives mostly by promoting attractive virus-cell interactions. To this end, VPs are either chemically modified on the outer sphere or non-covalently bound to additives.
Covalent attachment of additives to virus capsid yields stable and irreversible modifications. 104 However, the covalent attachment might interfere with transduction and deactivate viruses by shielding epitopes on the capsid surface necessary for adhesion. 105 Chemical modification of the virus surface requires mild conditions to maintain the integrity of VPs. 106 Due to the abundance of lysine in the capsid protein, 107 most bioconjugation methods are mild amine functionalizations at physiological conditions, e.g. using N-hydroxysuccinimide (NHS). 108 Other approaches apply azide-alkyne click chemistry 109,110 and utilize thiol-displaying bioengineered capsids for Michael addition. 111 Non-covalent attachment is simpler and generally obtained via attractive electrostatic interactions or incorporation in various scaffolds. This can result in different architectures such as coatings, [112][113][114] complexes, [115][116][117][118][119][120] capsules, [121][122][123][124][125] and matrices [126][127][128] as illustrated in Fig. 1. Table 2 provides a summary of these architectures and highlights their respective benefits and drawbacks.
Enhanced interactions between cells, VPs and additives can be obtained via various pathways.
For cationic delivery agents, binding is mainly driven by attractive electrostatic interactions. 129 This results in directed diffusion and colocalization of viral particles and cell membranes, thereby facilitating cell entry. Some materials can further enhance the fusion of viral and cellular membranes. 130 Cationic as well as anionic additives with high molecular weight can sediment with VPs onto cells in culture, which increases TE due to increased contact between VPs and cells. 131,132 VVs can also be immobilized on surfaces prior to cellular adhesion. 133,134 Chemically facilitated cell entry can also be achieved by manipulating the host cell itself. Especially small cytostatic molecules can affect viral infectivity and TE by intervening with cell cycle processes. 135 Applying delivery systems able to bypass receptor mediated cell entry can broaden tropism. For example, CAR is downregulated in cancer cells, which makes oncolytic Ad delivery challenging. 136 Delivery systems can improve addressing VPs to CAR negative cells and circumvent CAR-mediated endocytosis. 116,[137][138][139][140] A drawback of chemical delivery systems are cytotoxic reactions due to high additive concentrations. 114,141 Furthermore, non-degradable additives, e.g. high molecular weight polymers can result in unwanted accumulation and health risks in vivo. 142 Delivery systems Viral gene delivery systems including polymers, peptides, lipids, nanoparticles, and small molecules display diverse modes of action on a wide range of size scales (Fig. 2). The main focus of this review is to highlight materials for promoting viral gene delivery. the major issues with DEAE-D is cellular toxicity at high concentrations needed for efficient transduction, 143 which resulted in a declining interest for its use as a delivery agent for VVs.
Beside DEAE-D, the cationic polymer polybrene was heavily used as an enhancer of viral delivery in vitro in early days. 146 Due to its cost efficiency as well as its simple and safe handling it remains a popular additive, 147 e.g. for enhancing delivery of Ad to human mesenchymal stem cells (hMSCs) 148 and as an additive in ultrasound-enhanced RV delivery to the retina. 72 In general, it can be summarized that polycationic compounds like DEAE-D, polybrene, poly-L-lysine and protamine sulfate enhance TE, whereas polyanionic compounds like heparin, pyran or cyclodextrin modified with carboxylic groups inhibit TE and reverse the effect of polycationic additives, when combined. 146,[149][150][151] However, anionic dextran sulfate in certain concentrations could promote focus formation of Rous sarcoma virus and thereby slightly enhance TE. 149 In contrast to these findings, dextran sulfate inhibited TE of HIV-1 152 and neutralized enhancing effects of cationic additives. 149 The anionic polysaccharide chondroitin sulfate C also showed  unexpected increase of TE in combination with polybrene in vitro. This observation was explained by sedimentation of larger polymer-virus complexes on cells in contrast to free diffusion of unmodified viruses in the supernatant solution. 131,153 In order to yield VVs with improved safety profile, e.g. protect them from neutralizing antibodies and evade immune response, polymers have been coated 154,155 or covalently bound [156][157][158] to viruses. The safety of polymer-coated VVs has been increased by changing tropism and retargeting cells. 156 Further, natural and synthetic hydrogels from polymers have been investigated for spatially controlled VV delivery. 159,160 Amongst them macroporous hydrogels were utilized for longterm release of VVs, [161][162][163][164] and thermoresponsive polymer gels were applied as injectable systems. 127,163 A selected overview of general structural motifs of the polymers and a summary of all  polymers discussed in the following subsections are provided in Chart 1 and Table 3, respectively. PEG. Polyethylene glycol (PEG) is one of the most versatile polymers used in vast technical and consumer related areas due to its stability and biocompatibility. 165 For more than half a century, the pharmaceutic industry has employed PEG for shielding proteins from humoral immune response to prolong circulation time. [166][167][168][169] Consequently, viruses coated with PEG (PEGylated) were first developed to circumvent the immune response in vivo. An extensive review of PEG utilized for viral delivery was published in 2010. 170 Covalent attachment of functional PEG derivatives to Ad capsid proteins, first introduced by O'Riordan in 1999, 106 protected the virus from neutralizing antibodies 106,157 and displayed reduced clearance rates by Kupffer cells. 171 However, PEG-coated stealthy VPs are hindered in cell membrane interaction, which can be tackled by bifunctional multipurpose PEG linkers. Cell specific bioactive peptides attached to these PEG linkers introduced targeted high affinity interaction while showing low immunogenicity even in systemic administration. 172,173 Protection from neutralizing antibodies has been shown for PEGylated AAV at certain cross-linking ratios. 174 Interestingly, TE of VPs varied depending on the grafting efficiency of the activation agents for covalent attachment of PEG and their susceptibility to induce aggregation of VPs. 175 Genetic modifications of AAV resulting in expression of thiol groups on the capsid surface extended bioconjugation methods with thioether or disulfide bonds, e.g. for selective conjugation of NHS-functionalized PEG. 83 Moreover orthogonal conjugation of PEG derivatives can also be achieved by click chemistry, when genetically modified AAV capsids presenting azide moieties are used. 176 PEGylated Ad with low molecular weight of 5 kDa were successfully applied for accumulation in tumor tissue and showed enhanced permeability and retention (EPR) effect when injected intraveneously. 177 Noteworthy, in other studies, PEGylation of Ad with 20 kDa PEG instead of 5 kDa resulted in reduced liver transduction and hepatotoxicity. 178,179 PEGylation also shields Ad from pre-existing Ad antibodies. This is relevant for applications in vaccines or gene therapy due to possible pre-existing immunity against Ad in humans. 180 In vitro TE of PEGylated Ad was reduced compared to in vivo. This observations was traced back to higher cellular uptake with integrin and proteoglycan interaction, which is more strongly induced by pharmacodynamic force in vivo. 181 Beside neutralization of Ad by the serum, blood cells can also inhibit Ad activity after systemic administration. 182 Addressing this need, PEGylated Ad has been used to circumvent endothelial cell activation from blood cells and thereby improve TE and safety. 183 However, a crucial issue is the so-called PEG-dilemma. Concurrent to EPR effect, PEGylated viruses show lower cellular uptake and lower endosomal escape eventually leading to reduced TE. In order to overcome this problem, PEG was combined with other polymers as reviewed elsewhere for non-viral gene delivery. 184 Copolymers from PEG include spherical poly-DL-lactide particles, 121 pH-sensitive poly(L-histidine-co-L-phenylalanine), 185 Fig. 3). This polymer design aimed to overcome liver accumulation by using PEG 5 kDa, and further to electrostatically coat Ad by grafting PEG onto a high-molecular weight, branched PEI-cyclodextrin copolymer via matrix metalloproteinase (MMP) sensitive peptide linkers Chart 1 Overview of general structural motifs of selected polymers. (GPLGIAGQC). These peptides are cleaved in the environment of the tumor and uncover positive charge of PEI thus enhancing cellular uptake and Ad gene delivery. Thereby CDPCP avoids liver accumulation, inhibits Ad blood cell interaction and prolongs circulation time. 138 These advances achieved in stimuli-responsive targeted delivery have the potential to be a valuable tool for systemic administration of viruses, e.g. of oncolytic viruses to tumor tissue, in future clinic applications. However, despite extensive use of PEG as a delivery system, in the 2010s, unexpected immune response and accelerated blood clearance of PEGylated therapeutics were reported. 188,189 Consequently there is a need for alternative polymeric delivery systems. 190 PEI. Polyethylene imine (PEI), commercialized as jetPEI ® , is a commonly used transfection agent in non-viral gene delivery and enhances TE by assisting cellular uptake and endosomal escape. 191,192 The secondary and tertiary amines in the back-bone of the polymer yield positive charges at physiologic pH and promote attractive electrostatic interactions with anionic particles. After cell intake PEI can deliver its cargo directly into the cytoplasm by accelerated endosomal rupture without entering the degradative lysosomes due to its proton sponge effect induced buffering capacity and osmotic swelling at lower intracellular pH. [193][194][195] PEI can be applied in linear or branched forms for viral delivery. 196 One major drawback especially of high molecular weight PEI (25 kDa) is its cytotoxicity. 114,141 Attempts have been made to overcome this disadvantage by copolymerization. Grafting PEG onto PEI, 139,197 cross-linking with cyclodextrin, 198,199 introducing bioreducible disulfide groups 196 or diethylene glycol 200 improved toxicity, hemolytic activity, and enhanced transduction of Ad to CAR-negative cells. Lower immunogenicity was achieved through approaches such as layer-bylayer deposition of PEI with hyaluronic acid 201 or chondroitin sulfate 202 and by cross-linking PEI with cystamine. 203  Moreover cholesterol conjugated to PEI can facilitate cell entry via an energy-and endocytosis-independent membrane translocation pathway, originally known from cell penetrating peptides. 204,205 The amphiphilic combination of bile acids like deoxycholic acid and the cationic PEI efficiently transport cargo 206 and further enhance TE of Ad in CAR negative cells. 207 An elaborate approach of PEI assisted gene delivery was applied by Pan et al. for genetic manipulation of primary T-cell to target malignant tissue as new potent immunotherapeutics. In a stepwise process, they first labelled T-cells with azide groups by metabolic incorporation of azide-glucose onto the membrane, then adding PEI-dibenzocyclooctyl-complexed LV. Conjugation of azide-dibenzocyclooctyl via click chemistry resulted in colocalization of complexed LV with T-cells, which facilitate interaction and robustly enhance TE in T-cells (Fig. 4A). In vivo experiments with tumor-bearing mice displayed significantly longer survival times than the control group or polybrene-assisted delivery. 208 Delivery systems with spatially resolved gene expression have the potential to mimic the complexity of biological systems. An interesting example is branched PEI, which was functionalized with catechol groups and coated onto AAV. In this way, viral particles were obtained which could be placed and immobilized on tissue culture plates by a micropipette (Fig. 4B). Increased amounts of polymer coatings led to more stable immobilization resisting several washing steps and allowing to pattern viral particles. Thus, spatially resolved gene expression and cell manipulation could be achieved. 209 Recently, spatially defined transduction has been further simplified by coating poly(glycidyl methacrylate) copolymerized PEI as viral affine substrate. LVs were immobilized simply via electrostatic interactions and colocalized with HUVECs by incubation on this platform, resulting in enhanced TE and maintained cell functions (Fig. 4C). Further, patterned transduction and GFP expression could be generated by covering the substrate with a mask during fabrication. 210  In recent years, PEI has evolved from a simple polymeric additive to a popular element in copolymeric formulations for enhancing gene delivery, as copolymers can mediate inherent drawbacks of PEI such as high toxicity.
Polyesters. Poly(lactic-co-glycolic acid) (PLGA) is an FDA approved copolymer of lactic acid and glycolic acid and commonly used as microspheres for controlled release of drugs and in tissue engineering. 211 PLGA encapsulation of cargo is typically achieved by an emulsion process without the need for charge induced adhesion. 211 However, cationic stabilizers like chitosan and PLL facilitate encapsulation by inducing attractive electrostatic interactions between negatively charged cargo, such as VPs, and PLGA. 212,213 While Ad encapsulated in PLGA displayed greater cell viability and lower immunogenicity in vivo, it did not enhance TE. 155 The TE could be enhanced by PEGylation 214 before the encapsulation step and by surface modification of PLGA spheres with polysaccharides. 12 In a tissue engineering approach, LV or Ad was immobilized in a PLGA scaffold and ensured long-term transgene expression over a period of four weeks in vivo. LV or Ad were incorporated in the PLGA scaffold by consecutive lyophilization of both components together with sucrose. Additional fibronectin and collagen could further enhance binding but not TE for LV delivery. 133 Zhu et al. incorporated Ad in PLGA scaffolds for bone regeneration in vivo. The electrospun PLGA yielded a nanofibrous scaffold, which enabled spatially defined, long-term gene expression of Ad encoding hBMP2 to promote osteogenic differentiation. This technique was able to cover more than 80% of the bone defects that could not be repaired in the control groups within eight weeks. 215 Another electrospinning approach coated oncolytic vaccinia virus onto PLGA nanofibers to maintain their antitumor activity at the tumor tissue site. 216 Furthermore, melt-processing was reported as a solvent-and additive-free approach for fabrication of PLGA-VP scaffolds. This homogenous dispersed composite materials for prolonged VP release are created by heating up PLGA and VP. 217 Another example of polyesters is polycaprolactone (PCL) which is widely used as long-term implants in biomedical applications due to their good mechanical properties, slow degradability and biocompatibility. 218 In recent years, electrospinning techniques emerged as a promising process to create PCL fibrous structures for sustained and localized gene delivery. 219,220 In order to incorporate cargo to the fibers, coaxial spinning techniques can be utilized, in which an inner jet with cargo molecules and outer jet with polymer solution overcome the limitations of single stream electrospinning. 221 In one such approach, Ad was encapsulated in PCL in a core-shell fashion for subsequent porogen-mediated release. Pores of approx. 3 µm size were created by leaching out incorporated low molecular weight PEG particles. The fibrous scaffolds enabled efficient local TE and reduced macrophage activation to attached cells. 164 Similar results were obtained with PCL containing copolymers of polyester urethane urea and polyester ether urethane urea electrospun together with low and high molecular weight PEG and AAV to yield various fibrous nanostructures for sustained delivery to cardiac tissue over a period of 2 months in vitro (Fig. 5A). 222 Blending PCL with elastine-like pentapeptide (ELP) together with AAV resulted in nanofibrous scaffolds with adjustable degradability dependent on the polymer ratio for controlled AAV release. 223 Moreover, cysteine tagged proteins binding selectively to AAV, were attached to maleimide-displaying PCL microspheres or electrospun fibers and gave access to a therapeutic platform for intramuscular injection or subcutaneous implantation with reduced off-target (Fig. 5B). 224 Recently, poly(ε-caprolactone)-grafted poly(sodium styrene sulfonate) films immobilized rAAV and enhanced gene delivery to hard-to-transduce hMSCs in vivo, thus enabling less invasive, effective treatment of focal cartilage lesions. 225 PLGA and PCL were successfully applied as highly versatile platforms mainly as electrospun fibers and microspheres with controllable spatiotemporal gene delivery, especially considering applications in tissue engineering. Due to its biocompatibility and slow degradation rate both materials, PLGA and PCL has proven to be a reliable scaffold and a promising material especially for local, long-term gene delivery in vivo.
Poloxamers. Poloxamers, or Pluronics as a trade name, are commercially available, FDA-approved, thermoresponsive ABAtype triblock copolymers PEO-PPO-PEO consisting of two blocks of hydrophilic poly(ethylene oxide) (PEO) and one block of hydrophobic poly( propylene oxide) (PPO). 226 Poloxamers are sold in various block lengths and commonly used in pharmaceutical and cosmetic applications as surfactants with no safety concerns. 227 More recently, poloxamers have also been applied as gels for gene delivery, as they exhibit thermal gelation behavior at concentrations higher than 20%. 228 For example, suspensions of VPs cooled at 4°C can be gelated at physiological temperature around 37°C, which gives access to homogeneous, injectable, spatially precise viral delivery in vivo. 229 However, a potential adverse effect of poloxamer injection concerning the temporal accumulation of microclots in vessels and organs has been reported. 142 Several studies have shown enhanced TE of localized viral delivery by poloxamer 407 (PEO 101 -PPO 56 -PEO 101 ) amongst others to vascular smooth muscle cells in vitro, 230 to arteries in vivo, 231 to the central nervous system, 232 to solid tumors in vivo, 229 and for treatment of spinal cord injury. 160 Rey-Rico et al. applied a series of poloxamers for enhanced AAV delivery to hMSCs even in the presence of host cell receptor binding inhibitory heparin or VP neutralizing antibodies. Prior to gelation, the VPs were encapsulated in poloxamer micelles to shield from adverse conditions, and cell viability was maintained to 100% over a period of 21 days during gene transfer in culture. 233 Long-term expression of AAVs to hMSCs have previously been reported for fibrin or RAD16-1 encapsulated viruses for shorter periods compared to poloxamer encapsulation. 234,235 Linear poloxamer PF68 and four branched poloxamer T908 encapsulated rAAV in a micellar architecture and enhanced spatiotemporal gene delivery efficiency to osteoarthritis chondrocytes in vitro. 236,237 Recently, treatment of cartilage damage was applied in a clinically relevant in vivo minipig model with rAAV delivered by poloxamer 407 (Fig. 6). Controlled release of the therapeutic SOX9 from the poloxamer gel improved repair of full-thickness chondral defects. 238 To further enhance adhesiveness of poloxamer 407 for local viral delivery to organ surfaces, it was blended with >1% polycarbophil, a polyacrylic acid cross-linked with divinyl glycol. Addition of polycarbophil changed the sol-gel transition temperature and led to higher adhesiveness due to numerous carboxylic groups, which can easily form bonds with surrounding molecules. Spatially resolved and stable Ad delivery to heart tissue was shown in vitro and in vivo without having adverse impact to TE or cell viability. 239 Poloxamer 338 (PEO 141 -PPO 44 -PEO 141 ), also traded as synperonic F108, enhanced LV delivery in difficult-to-transduce T-cells more efficiently than polybrene. In combination with polybrene the TE was further elevated, which was explained by the distinct modes of each adjuvant, polybrene compensating electrostatic repulsion and poloxamer 338 fluidization the membrane. 240 More recently, poloxamer 338 was commercialized as LentiBOOST™, and applied for LV delivery to CD34+ stem cells, 241-243 CD4+ and CD8+ stem cells, 244 and T-cells. 245 Enhanced TE, non-toxicity and clinical relevance were emphasized in all of these studies. The evolving progress in poloxa-mers as adjuvants for viral delivery in clinical application holds great promise for improved gene-based treatments.
Dendrimers. Dendrimers are branched, radially symmetric molecules with a high density of functional surface moieties,  monodisperse size and internal cavities, which makes them an attractive nanomaterial as a vector carrier. 246 A general overview of various dendrimers used in non-viral gene delivery can be found elsewhere. 247 Early works focused on inhibiting virus infections 248 or were used to control assembly 249 of viruses without any relation to gene delivery.
Polyamidoamine (PAMAM) is a common cationic dendrimer frequently used in non-viral gene delivery 250 and was applied for the first time to enhance TE of Ad by Vetter et al. 140 An epidermal growth factor receptor (EGFR) targeting peptide sequence was coupled to the dendrimer via a PEG-linker and coated on Ad, which resulted in increased specific infection to EGFR overexpressing tumor cells. The PAMAM complexed Ad showed lower cellular toxicity and higher TE than branched PEI. 140 EGFR targeting antibodies attached to PEGylated PAMAM also showed enhanced TE of Ad and tumor suppression in vivo. 251 Further, polyphenylene dendrimers (PPD) were applied for viral delivery. PPD have amphiphilic properties forming a rigid globular architecture in solution with functional end groups organized like a shell in the periphery. 252 These features make PPD a promising protein-mimicking drug delivery agent. 253 Recently, Wu et al. reported PPD3 with engineered amphiphilic surface patches mimicking a protein corona, which enabled non-electrostatic interactions with Ad. The capsid proteins were complexed by PPD due to amphiphilic interactions, which resulted in enhanced TE to CAR negative cells and protection from neutralizing antibodies and the coagulation factor X. 116 Further remodelling of PPD3 led to one amphiphilic dendron branch, which was one quarter of the original PPD3 size (Fig. 7). This minimized dendron branch allowed Ad binding and non-covalent post-modification of viral capsids while maintaining advantageous properties of PPD3. 254 One disadvantage in using dendrimers is their laborious synthesis. Branched polymers carry a large number of functional groups, similar to dendrimers, but are often easier to synthesize. One example is the cationic copolymer EGDE 3,3′ containing the monomer ethylene glycol diglycidyl ether (EGDE) and 3,3′diamino-N-methyl dipropylamine (3,3′), which induced higher TE of Ad to bladder cancer cells compared to the transfection reagent PEI. 255 Polysaccharides. Biomaterials from polysaccharides provide a versatile and biocompatible tool for clinical applications. Most polysaccharides used for viral delivery are hydrogels building a matrix for spatiotemporal release of VVs. Matrixmediated viral delivery was recently reviewed elsewhere. 256 The following sections mainly cover saccharides in hydrogel applications.
Alginate is a linear copolymer consisting of β-D-mannuronate and α-L-guluronate and is commonly used as a hydrogel in food additives or pharmaceutical applications. Even though unmodified alginate hydrogels cannot interact specifically with mammalian cells, 257 a lot of progress has been made using such gels in controlled VV release in recent years. Ad encapsulated in alginate circumvent immune response in vivo 258 and augment long-term oncolytic Ad infection to tumor cells. 259 Distinct hydrogel-rAAV capsules were formed by tuning the composition and cross-linking temperature for alginate and poloxamer 407 containing systems. All of these rAAV capsules showed enhanced targeted delivery to hMSCs. 122 The spatiotemporal release kinetics of VVs can be stunted or enhanced by hydrogel physiochemical properties. 260,261 For example fabrication methods determine hydrogel mesh size, matrix affinity interactions and degradability and thus result in varying release kinetics for different sizes of VVs. LV delivery from CaCO 3 or CaCl 2 gelated alginate microcapsules generated by a microfluidic technique correlated with mechanical properties like hydrogel network mesh size and degradation rate and was controllable by the gelation method. 261 Moreover, low molecular weight alginate gels show faster degradation and release of LV, compared to more sustained release from high molecular weight alginate. 260 On-chip fabrication of alginate microgels gave access to blends of adjustable alginate formulations in a bottom up approach (Fig. 8A). Depending on the composition of different alginate molecular weights the degradation and release time of LV could be controlled in vivo. 262 Building on these results, Madrigal et al. reported that physical properties of various formulated alginates impact AAV and LV delivery to a different extent (Fig. 8B). LV release was tuned by initial strength and degradation rate of alginate gel, whereas AAV delivery remained unchanged independent of formulation. This result was interpreted as release of AAV by diffusive transport, whereas LV release is mainly controlled by degradation rate of alginate, which may be due to mesh/VP size relation. Consequently fast degradable alginate gels lead to higher TE for both AAV and LV. 263 Mannose receptors were highly expressed in liver tissue and endocytosis-mediated cell entry is enhanced in the presence of mannose. 264 In order to target hepatocellular carcinoma, polymannose was covalently attached to Ad surface either by reductive amination 265 or by oxidation with sodium periodate. 266 The polymannose-Ad conjugate showed enhanced gene delivery to hepatocellular carcinoma cells both in vitro and in vivo. 266 Chitosan is a non-toxic, biodegradable cationic polymer, that shows neuroprotective effects after spinal cord injury by sealing nerve cell membranes 267 and promotes peripheral nerve regeneration. 268 Cross-linked with β-glycerol phosphate, chitosan yields compact fibrous hydrogels with increased charge density and binding to anionic particles as well as long-term gene expression over a period of seven days of encapsulated LV to dorsal root ganglia neurons. 127 Furthermore, chitosan has been used to replace the function of the viral envelope in non-infectious murine leukemia virus (MLV), which led to increased infectivity and transduction. 269 Hyaluric acid (HA) is a highly abundant linear polysaccharide in the extracellular matrix. HA applied as an injectable in situ forming scaffold can generate macroporous structures which are attractive vehicles for localized long-term release of viral particles. In a recent report, HA scaffolds of various pore sizes for LV delivery in mice mammary fat were compared with each other. Void spaces in HA scaffolds were created by different fabrication techniques. Nanoporous structures were achieved by cross-linking HA particles with PEG precursors, while macroporous architectures were created through in situ assembly of HA particles with PEG particles or enzymatic degradation of included PEG particles. Open, macroporous HA-PEG hydrogels displayed increased host cell infiltration and yielded higher TE compared to nanoporous hydrogels. 162 Cyclodextrins (CD) are composed of 6-8 (α-γ) cyclic arranged glucose subunits forming a hydrophobic interior and a hydrophilic exterior toroid-shaped oligosaccharide, which is commonly used in pharmaceutical applications for drug delivery. 270 CDs have been applied as hydrogels in combination with other polymers 138,271 or as supramolecular linkers 272 for gene delivery as recently reviewed elsewhere. 273 Examples include Ad delivery to tumor microenvironment with a responsive polymer design containing PEG, PEI, MMP-sensitive peptide and β-CD. 138 Further, polypseudorotaxane gels based on either HA or chondroitin sulfate combined with α-CD were used to encapsulate and release rAAV to hMSCs. α-CD enhanced the viscoelasticity and storage modulus of the gels at physiological temperature and prolonged the permanence at the application site. 271 CD were also covalently attached to TMV surface in a supramolecular strategy to enable facile host-guest interactions with adamantyl moieties of imaging agents or chemotherapeutic drugs. 272 In order to overcome anionic surface charges of some polysaccharides, modifications and combination approaches with other polymers have been made. For example, cellulose nanocrystals were surface-modified by atom-transfer radical polymerization with poly(N,N-dimethylaminoethyl methacrylate) to yield a brush-like cationic polymer (Fig. 8C). This polymer showed high-affinity virus binding. 274 Bioreducible, branched polyaminoglycosides involving the antibiotic aminoglycoside tobramycin were proposed by Xu et al. as a transfection reagent. 275 Recently, they reported, LVpolyaminoglycoside complexes, which efficiently induce cell apoptosis to glioma cells by facilitating cellular uptake via endocytosis pathways. 276 Polysaccharides are highly suitable biopolymers for viral gene delivery, mainly as hydrogels that adapt to the tissue environment. Release of VPs is achieved by degradation into non-toxic components. Combination of polysaccharides with polymers and functional molecules can further expand the range of material properties and influence controllable and efficient delivery of VPs in future applications.
Miscellaneous. In recent years, several additional polymers have been explored as VV delivery agents. In this section, miscellaneous emerging polymeric delivery systems are briefly introduced.
Several examples of hydrophilic polymers with similar stealth properties to PEG have been reported. For example, poly(2-oxazoline)s are thermoresponsive, hydrophilic polymers which gained increasing attraction for biomedical applications. 190,277 Recently, hepatitis B-like viral particles grafted with poly(2-ethyl-2-oxazoline)s were reported to reduce antigenic reaction. 278 Similarly, poly(N-(2-hydroxypropyl)methacrylamide) ( pHPMA) was conjugated through lysine residues on Ad surface and was able to shield from neutralizing antibodies, enhanced plasma circulation, decreased hepatotoxicity and indirect accumulation in tumor site. However, coating with pHPMA prevented efficient cellular uptake and reduced TE. 156,158,279 Francini et al. applied a new conjugation strategy with pHPMA bearing diazonium reactive groups, which can be bioconjugated onto a variety of functional amino acid residues of oncolytic Ad resulting in more efficient coupling and dense coverage of surface. While immunogenicity of conjugated VPs is low, TE is severely reduced due to low cellular uptake and delayed unpackaging of the vector. This study illustrates the necessary balance between efficient screening of the virus from being recognized by the immune system and sufficient cellular uptake in order to achieve effective transduction. 280 To take advantage of benefits from different systems, hybrids consisting of polymers and peptides have been investigated for enhancing TE. For example, arginine grafted onto poly(disulfide amine) were able to enhance TE, while being bio reducible and less immunogenic, when coated onto Ad. 114,281 AAV in hydrogels from PEG incorporated with poly-L-histidine showed ratio-controlled and pH-dependant swelling and TE (Fig. 9A). 282 PEG diacrylate matrices (PEGDA) blended with PLL can improve long-term, localized and efficient LV transduction when implanted in vivo. 283 Further, Kwon et al. introduced elaborate viral/non-viral chimeric systems by siRNA or DNA encapsulating polyketals assembling in a core-shell structure around AAV. This design enabled simultaneous gene transduction in a stimuli-responsive fashion by using of polyketals that are degraded in the acidic endosomal environment. Chimeric systems are promising platforms for obtaining syner-gistic therapeutic effects by simultaneous expression and silencing of multiple genes. In cancer therapy, for example, simultaneous upregulation of pro-apoptotic mediators by AAV delivery and silencing of pro-survival genes by siRNA, can result in significantly more effective treatment. 284,285 Beside classical polymer systems, DNA-aptamers have also been utilized for targeted gene delivery. DNA-aptamers were covalently attached to viral capsid and selectively targeted Jurkat T cells and delivered cargo through an endocytic pathway. 286 DNA aptamers can further improve biocompatibility of viruses by shielding them from neutralizing antibodies and enhancing in vivo circulation rate. 287 Reducible disulfide linkages were utilized to covalently attach AAV to multiple DNA-aptamers, which were cleaved by intracellular glutathione and facilitate release of AAV in the cell, thereby enhancing TE. 288 Polymers can also be used as 2D coatings to promote transduction by colocalization of viruses with host cells. So-called substrate mediated delivery was enabled via AAV capturing adhesive catecholamine surfaces (Fig. 10A). 289 Adhesive polydopamine-coated substrates were further improved in an simple upside down arrangement to adhere inverted quasi spherical droplets containing human neural stem cells (hNSCs) and AAVs (Fig. 10B). TE was enhanced by shortened path lengths and increased contact frequencies between cells and vectors due to the large contact angle of droplets on polydopamine coated surfaces. 290 Several hydrophilic polymers have been applied as 3D materials, namely hydrogels, often for slow release of VPs. The biodegradable multiblock sulfamethazine and PEG-containing polyurethane (PUSMA) exhibit pH and thermoresponsive behavior and was used as an injectable hydrogel for in vivo oncolytic Ad delivery. The sol-gel transition occurred below pH 8.0 through a hydrophilic to hydrophobic transition leading to the formation of a porous hydrogel network. This hydrogel enabled spatiotemporal Ad delivery to injection site under physiological conditions (Fig. 9B). 163 An interesting example for the rAAV delivery to the cornea used hydrogels from a copolymer of hydroxyethyl methacrylate (HEMA) and aminopropyl methacrylamide (APMA) which can be worn as contact lenses (Fig. 9C). This transparent hydrogel network allowed high vector loading and controlled, long-term gene expression over a period of 14 days while being able to correct refractive errors. 291 Microgels from vinyl ether acrylate-functionalized poly(vinyl alcohol) (PVA-VEA) and thiolated PVA-VEA were fabricated by a microfluidic technology and Michael-type cross-linking reaction to yield pH-degradable injectable spheres for efficient Ad delivery to tumor sites. The antitumor treatment could be reinforced by the addition of the bromodomain inhibitor JQ1. Accumulation of oncolytic Ad at the tumor site was enabled by pH-dependent controlled release from microgels. This combination approach paves the way for treatment of tumors by synergistic viral, chemo and immunotherapy as a promising system in clinical applications. 292 Polystyrene coated with methyl methacrylate and divinylbenzene were formed into nanocups <500 nm by a template and applied as cavitation mediated carrier for oncolytic VV. Physical stimuli, e.g. ultrasound after intravenous injection resulted in enhanced transport and antitumor activity to treatment site. 293 Several new polymeric systems and composites are emerging. Careful choice of the polymer and conjugation strategy allows the user to tailor pharmaceutical properties such as circulation time, controlled release of viral cargo and targeting of cells or tissues for next generation gene therapy.

Peptides
Due to their natural abundance and their ability for interactions with cells and viruses, bioactive peptides are highly interesting auxiliary agents for VV delivery. 296 Tailor-made peptides can be easily produced in large scale, are biocompatible and biodegradable.
Peptides applied for non-viral gene delivery were summarized recently elsewhere. 297 When used as enhancers for viral gene delivery three different classes of peptides have been applied so far: cell penetrating peptides (CPP), fibrils formed from self-assembling peptides and proteins.
The formation of peptide-virion complexes is typically achieved either by attractive electrostatic interactions between negatively charged viral particles and positively charged peptides or by bioconjugation of the respective peptide to the capsid. With the large structural variety offered by peptide sequences, it is not surprising that many different types of peptides have been reported as promoting viral gene delivery. Table 4 provides an overview of the reported peptides, their sequences, and physicochemical properties. Furthermore, the types of VVs that have been reported in combination with these peptides are highlighted.
CPPs. Cell penetrating peptides (CPPs), also called protein transduction domains (PTD), typically contain between 5 and 30 amino acids, have an overall positive charge and can facili-    tate efficient entry into cells. CPPs can exhibit various secondary structures from random coil to α-helix, 298 and typically have a large amount of basic amino acids like arginine or lysine and have an amphiphilic or hydrophilic character. CPP motifs are widespread in Nature, e.g. in heparin-, RNA-and DNA-binding proteins, signalling peptides or viral proteins and allow receptor independent cell-entry. 299 Since the discovery of the first CPP derived from HIV-1 binding protein Tat in 1988, 300,301 more than 1000 CPPs predominantly from natural origin or found by phage display are known today. 302 Their ability to transport variable cargo across cellular membranes has made CPPs a facile tool for the delivery of DNA and RNA, liposomes, nanoparticles, proteins, and drugs as reviewed elsewhere 303,304 and of viral nanoparticles for efficient transduction as comprehensively reviewed. 305,306 In this segment, we highlight the most important developments in CPPs for facilitating transport of VP into target cells.
The most straightforward and easy way to obtain CPP-virus complexes is by co-incubation, making use of electrostatic interactions between positively charged CPPs and negatively charged VP. This method can be applied independently of the virus type, and ratio of CPP to VP can be easily adjusted in solution. However, the formation of CPP-VP complexes cannot be controlled, there is batch-to-batch variability and the highly positively charged CPP-VP complexes are prone to aggregation in physiologic conditions, for example, in the presence of electrolytes or serum (Fig. 11). 307 A more stable but demanding approach is to covalently connect CPPs to the vector capsid. Early studies have demonstrated both approaches: the utilization of electrostatically bound Pen and Ad complexes to facilitate viral gene transfer in muscle cells 308 and covalently bound Tat-Ad conjugates for delivery to tumor cell lines. 309 By covalently attaching Tat to exposed lysine residues of the vector capsid via an MHS linker, transduction efficiency was further improved. 310 Similar observations were made for Tat, Pen, R8 and Pep1 when they were covalently attached to PEGylated adenoviruses (Fig. 11). 311 Coating of CRRR-co-PEG-co-CRRR onto an oncolytic Ad (Fig. 12A) enhanced transduction in tumor sites with a longer blood circulation time and lower liver sequestration was achieved. 312 In addition to CPPs derived from natural peptides, screening for suitable sequences by phage display has become a powerful method to discover new CPPs for enhanced transduction and targeted virus delivery. 313,314 It was further possible to broaden the tropism of Ad by gene transfer to otherwise non-transducable CAR-negative cells. To this end, the surface knobs were modulated with Tat peptides, 137,315 Tat peptides were attached to surface bound lysine residues 316,317 or simply incubated in solution. 318 Other examples for hard-to-transduce cell types that are successfully targeted with CPPs include Ad delivery to resistant stem cells and various cancer cells which was efficiently achieved by addition of arginine-rich HP4 derived from herring protamine. 119 Furthermore, by decorating CPPs onto capsids via hydrazone chemistry, plant viruses like CPMV and TYMV were able to transduce otherwise non-infectable mammalian cells. 319,320 Specific CPPs capable of crossing the blood brain barrier (BBB), can facilitate the delivery of CPP virus-complexes to the central nervous system after systemic application (Fig. 12B). 321 In an interesting approach AAV containing a brain derived neurotrophic factor fused with Tat were delivered intranasally to the central nervous system to act as an antidepressant in mice. 322 Frequently used CPPs for VV delivery are various Tat peptide fragments, oligoarginines, and penetratin (Pen). These peptides show different transduction enhancing properties depending on the viral particle, host cell, and CPP concentration. 318 This difference in enhancement is believed to result from structural properties of the CPPs as well as their respective cellular entry mechanism. In general, various endocytic and non-endocytic cell entry pathways are controversially discussed in the literature. 323,324 Regarding the structure, one requirement for the electrostatic stabilization of the virus peptide complex, as well as adhering to and crossing the negatively charged lipid bilayer of cells, is a high amount of positive charges in the peptide. 318 Among peptides with positive charge those rich in arginine showed greater membrane permeability than CPPs with high amount of other cationic amino acids like lysine, histidine or ornithine. 325,326 Beside the charge, hydrophilicity can also influence interaction with the membrane. It is believed that the higher performance of hydrophilic peptides like Tat and oligoarginines is due to stronger interactions with heparan sulfate proteoglycans of the cell membrane, whereas amphipathic peptides like Pep1 interfere with electrostatic binding of proteoglycans, thus resulting in lower transduction efficiency. 318 Interestingly, a higher CPP concentration for VV delivery did not necessarily lead to a linear increase in transduction efficiency as shown for Tat, Pen, oligoarginine and Pep1. 316,318 Chirality also significantly affects cell permeability. A 9-mer of poly-D-arginine showed five-fold higher cellular uptake than poly-L-arginine. 327 Mechanistic pathways for the TE of AAV-CPP complexes were investigated by delivering AAV to non-permissive cell lines and blocking specific receptors. The tested peptides Pen, Tat-HA2 and LAH4 facilitate energy dependent and independent endocytosis as well as receptor-mediated pathways for the internalization of AAV. 328 Further, Tat-Ad promoted cellular uptake via heparane sulfate receptors on the membrane surface, while the oligoarginine adenovirus conjugate R8-Ad was more dependent on chondroitin sulfate B receptors. This lays the foundation for CPP-dependent virus delivery. 316 In a comparative study, TYMV was bioconjugated to Tat, R8, Pen or Pep-1. Improved efficiency was observed for Tat, R8 and Pen, whereas Pep-1 showed no change in transduction. This was traced back to different internalization routes and distributions of CPP-virus complexes in the cytoplasm and visualized by confocal images (Fig. 12C). 320 Recently, Váňová et al. reported further mechanistic explanations for different TEs by investigating the influence of various CPPs on the activity and stability of VPs. They found that KH27K, FUSO, R8 and LAH4 affected the stability of VPs in different ways. KH27K promotes the aggregation and enlargement of VPs, while LAH4 destabilizes VPs but still enhances infection by concentrating them onto the cell surfaces (Fig. 13A). 329 This study provides an example for enhanced TE via virus disassembly, which was suggested in an earlier report covering Pen, Tat and R8 311 and provides the basis for further explanations of observed differences between CPPs. 320 Peptide fibrils. The intrinsic ability of some peptides to selfassemble into higher order structures allow for numerous applications as functional biomaterials. 330,331 In the last ten years, peptide fibrils caused a paradigm shift in viral gene delivery from using mostly spherical particles to anisotropic, fibrillar nanocarriers, due to their convenient handling and high TE.
In contrast to CPPs, fibrillar peptides typically have a distinct secondary structure and cannot necessarily cross cell membranes. Enhanced viral gene delivery is achieved by colocalization of electrostatically complexed peptides and viral particles with the cellular membrane. Due to the high aspect ratio and rigidity of fibrils it is assumed that they cannot adapt to and cover virus surfaces thoroughly, thus creating excess positive charges in the virus-fibril complex for electrostatic interaction with cell membranes. 332 The most thoroughly investigated fibrils for viral gene delivery are amyloids. Amyloid fibrils are highly stable and rigid, which makes them interesting materials for applications where long-term stability or tolerance of harsh conditions are required. For a long time, amyloid-forming peptides have exclusively been associated with diseases like plaque formation in Alzheimer's disease. 333,334 However, these structures were also found in non-pathogenic contexts as functional amyloids, for example on bacteria surfaces 335 and in seminal fluids. 336 In the latter case, prostatic acidic phosphatase (PAP), a semen derived enhancer of virus infection (SEVI) with two distinct regions PAP  and PAP  , and semenogelins (SEM) was found to form amyloidal structures and enhance HIV-1 infection. [336][337][338] These very first findings sparked further research in amyloid peptides for viral gene delivery. Transduction enhancing peptides can also be found in virus envelopes. The optimization of the glycoprotein fragments gp120 [417][418][419][420][421][422][423][424][425][426][427][428] (EF-C, commercialized as Protransduzin) and gp41 671-682 (P13 and P16) from the HIV-1 envelope and transmembrane protein, respectively, form small amyloid fragments that assemble into cationic nanofibrils of several hundred nanometers in length and a few nanometers in diameter (Fig. 13B). These short fragments showed higher TE compared to Tat, polybrene, DEAE and SEVI in various cell lines including difficult to transduce TZM-bl cells, while being cost effective and convenient in handling. 118,339 Fluorescent dyes coupled with free amino groups in EF-C have been introduced as a tool to study plasma stability and in vivo biodistribution while maintaining the structural and functional properties of non-labeled EF-C. 340 Point mutations in the peptide sequences had a great impact on TE as shown for P16-D, where a substitution of aspartic acid with alanine resulted in increased activity. 341 Sequence variations of gp120 [417][418][419][420][421][422][423][424][425][426][427][428] yielded the 6-mer DPF1 and longer sequences EP2 and EP3 which accelerate amyloid formation of SEVI and SEM and enhanced TE. [342][343][344] The fibrillar structure is necessary for enhanced VV delivery (Fig. 13C) 345 as shown by the following example: addition of epigallocatechin gallate, a main compound of green tea, which inhibits amyloid formation resulted in lower TE (Fig. 13D). 344 Amyloids can also help to broaden the tropism of viruses. For instance HIV-1 was able to transduce in hardly infectable CD4 negative cells with the addition of amyloidal CD4bs-M peptides. 346 Moreover, the cofibrillation of amyloid α-Syn with positively charged polymers like poly-L-lysine or chitosan enhanced TE of amyloid fibrils as recently reported by Maji and coworkers. The authors suggested that these positively charged additives increased electrostatic interactions and local density of virions on cell surface, thereby facilitating the transduction (Fig. 13E). 117 Besides using derivates of naturally occurring amyloids for vector delivery, amyloids can also be computationally designed as shown for KK and KY, which enhanced DNA delivery to mammalian cells by forming DNA-amyloid complex and overcoming charge repulsion, as discussed for VVs. 347 Since synthetic amyloid fibrils have so far only been applied in ex vivo studies, the question of fibril stability and degradability in vivo remains open.
Hydrogels formed from fibrillar peptides are emerging as VV delivery scaffolds, due to their simple handling and spatiotemporal release of viruses. For instance, Fmoc-SAP derived from the epitope IKVAV enabled localized delivery of LV after implantation into the central nervous system of mice. The modification of the sequence with an additional lysine at the C-terminus increased electrostatic interactions and immobilized the VP to obtain focal gene delivery to the site of injection (Fig. 13F). 348 The peptide hydrogel RAD16-1 (commercialized as PuraMatrix™) is frequently used in cell culture. It displays favourable nano-structural and biomechanical properties and promotes the proliferation of various mammalian cells. 349 Rey-Rico et al. reported RAD16-1 hydrogels for durable genetic modifications to stem cells by enhanced localized AAV delivery over a period of 21 days in vivo. 234 The LAH4 peptide family has attracted growing interest in recent years as a DNA transfection reagent as well as for VV delivery. 328,350,351 One commercialized derivative of the LAH4 family is Vectofusin-1. 351 This 26-mer cell penetrating peptide is the first transduction enhancer with α-helical fibrillar structure. Studies have shown enhanced TE of Vectofusin-1 compared to other delivery agents such as Tat, Pen, LAH4 derivatives, KH27K, R8, FUSO, polybrene, and retronectin. 328,[351][352][353][354][355] Interestingly, a variation in the histidine sequence order or peptide length resulted in a significant change of bioactivity. A minimum length of 21 amino acids of the LAH4 peptide family was found to be necessary for successful vector delivery. 355 Polypeptides. Polypeptides for VV delivery are mainly derived from natural sources and are typically used as scaffolds for enhanced delivery by colocalization.
Protamine sulfate is an early examples for viral transduction enhancers, originally derived from salmon sperm and clinically used for treatment of heparin overdose. This substance is actually a mixture of several polycationic peptides and facilitates virus-cell interactions by charge neutralization similar to other enhancers like polybrene, DEAE dextran or poly-L-lysine. 356,357 In comparative transduction studies of LV delivery to CD34+ stem cells, protamine sulfate showed a TE similar to Vectofusin-1 and higher than that of Retronectin. 243 TE could be enhanced even further by combining protamine sulfate with chondroitin sulfate. 358 Retronectin is a 574 amino acid long recombinant polypeptide based on the fibronectin fragment CH296. It is one of the first commercially available enhancers for VV delivery and has already been tested in clinical trials. [359][360][361] A typical workflow for transduction involves the coating of Retronectin on a solid surface, the addition of viral particles and cells followed by spin centrifugation. 362 To overcome the limitations of the laborious preparation on surfaces and the necessity of centrifugation, various alternative approaches have been devel-oped. The coating of epoxy-modified paramagnetic beads with Retronectin enables the effective capture of retroviral particles from solution and provides access to remote-controllable transduction. 363 In vivo LV delivery to stem cells via intra-bone marrow injection in mice showed enhanced TE when Retronectin was co-injected with the virus. 364 Poly-L-lysine (PLL) is a popular additive in cell cultures for reinforcing adhesion of cells to culture dish. It is used as short oligomers up to polypeptides of several 100 kDa and can either be covalently attached to virus surface or simply coated on surfaces. [365][366][367] PLL has been investigated as a replacement for polybrene in viral delivery by creating the block copolymer PEG 12000 -PLL 48 which was coated on RV through electrostatic interactions. In contrast to polybrene, this copolymer augments RV transduction to delicate primary cultured brain cells without cytotoxic effects. 368 Further optimization of PEG-PLL compositions were made by functionalizing four-arm PEG acrylates with PLL of different molecular weights ranging from 1-70 kDa. Increased TE was observed for increased molecular weights of PLL. 369 Moreover, PLL has been applied as a mimic of the viral envelope and enhanced cellular internalization and transduction of virus-like particles. 367 Recently, modifications of PLL 120 with p-toluylsulfonyl arginine resulted in change of conformation from random coil to α-helix, which gave higher DNA-transfection rates due to higher cell-permeability. 370 Layer-by-layer depositionreferred to as protein surface precipiation hereof PLL and dextran sulfate sodium salt were applied onto Ad surface to obtain nanospheres that protect Ad from antigenic reactions and thereby enhanced TE (Fig. 14A). 371 These recent findings highlight the importance of material deposition techniques and conformational structure for viral gene delivery.
Enhanced viral delivery with spatiotemporal control can be achieved by fibrous protein scaffolds, such as fibrin. For example, when VPs were incorporated in fibrin, a local, longterm release of Ad, 372 AAV 235,373 and LV 374 in vitro and in vivo was observed at the injection site up to 14 days. Fibrin scaffolds could be further improved by the addition of hydroxyapatite nanoparticles, resulting in stronger trapping as well as slower release of VPs from scaffolds 374 (Fig. 14B) and gels. 126 Vupputuri et al. reported that protein impurities in cell culture can lead to increase or decrease of viral TE. By carefully adjusting the amount of protein added to viral particles, they could improve TE of polymer-virus complexes. Proteins bound to VPs can increase TE due to sedimentation of heathier particles, however at the same time cellular uptake pathway is changed by larger VPs thus protein addition has to be carefully balanced. 132 This observation have far-reaching consequences for comparability of studies concerning TE, when taking batch-to-batch variability of culture medium and VP purification into account. For example, proteins from the human serum can also augment AAV transduction. Cationic human serum albumin (cHSA), an abundant endogenous transporter protein in blood, increased transduction of an in vivo hemophilia B mouse model fivefold. 375  genicity. 376 Combinations of HSA, low density lipoproteins (LDL) or transferrin yield AAV-serum protein complexes which enhanced transduction to liver in vivo, prevented binding of AAV to other proteins, and suppressed nonspecific binding to cells. 377 Finally, site-specific antibody conjugation to TYMV, CPMV and bacteriophage via azide-alkyne click chemistry was reported by Park et al. (Fig. 14C). 378 These examples highlight that natural proteins can efficiently enhance viral delivery. However, concerns of potential immunogenicity of xenogeneic materials exist. 379 Natureinspired, synthetic silk elastin-like protein polymers (SELPs) may circumvent this problem. SELPs mainly consist of repeating amino acid blocks based on the typical silk (GAGAGS) and mammalian elastin (GVGVP) motifs and were first reported for Ad release in cancer gene therapy in 2004. 380,381 SELPs showed enhanced, local, long-term Ad delivery up to four weeks in various studies. 382,383 In a comparative study with poloxamer 407, SELPs showed promising results with a superior TE, lower toxicity and immunoreactivity. 384 Furthermore, collagen, an abundant fibrous protein in the extracellular matrix, showed long-term localized gene expression in vivo up to 4 weeks when it was co-gelated with LV. 126 Organic-inorganic hybrid systems, such as collagen hydrogels combined with hydroxyapatite nanoparticles retain LV over a period of four weeks in vivo and increase local delivery efficiency even more. 126 Moreover, gelatin gels, which consist mainly of hydrolysed collagen, were also applied for viral delivery. Gelatin sponges and Ad were functionalized with biotin to link them after avidin addition. The virus tethered in a virus-biotin-avidin-biotingelatin arrangement showed enhanced spatiotemporal TE in vivo after implantation for bone regeneration (Fig. 14D). 385 Local long-term gene expression was also shown for oncolytic Ad delivery from gelatin gels in vivo up to 20 days. These gels could further protect Ad from immune response and prevent unwanted delivery to healthy sites. 386

Lipids
Lipids are amphiphilic molecules typically consisting of a hydrophilic head group and a hydrophobic tail. Lipids are the main component of cellular membranes, virus envelopes and extracellular vesicles, which are Nature's endogenous nanocarriers to deliver biological information from cell to cell. 387 The unique ability of lipids in aqueous media to form bilayers in a vesicular structure (liposomes) and spherical monolayers (micelles) enables them to encapsulate both hydrophilic and hydrophobic cargo. 388,389 After encapsulation the cargo is protected from degradation as well as immune reaction 390 and can further address specific targets by functionalization at the liposome's outer sphere. 391 Since their first discovery in the 1960s, 392 liposomes have been extensively used as delivery agents in clinical trials and pharmaceutical industry. 393,394 In non-viral gene delivery, lipids are currently the gold standard. For example, liposomes were applied as DNA and RNA nanocarriers 395,396 as well as mimics of the viral envelope for gene delivery. 397 Recent reviews on non-viral application of lipids for gene delivery can be found elsewhere. 398,399 Lipids for viral gene delivery, which are discussed in the following section are summarized in Table 5.
Formulation. In viral gene delivery, the most frequently used lipids (Chart 2) are cationic phospholipids (e.g., DOTAP, DOSPA and DOTMA), zwitterionic helper lipids (e.g., DOPE) and sterols (e.g., cholesterol (Chol)). 400 The internalization efficiency into cells is highly dependent on the formulation of lipid mixtures. 388,400 Usually the negatively charged viral particles are encapsulated in liposomes and subsequently taken up into cells. 150,400 For example, commercialized transfection agents like Lipofectamine™ and Lipofectin™ contain the mixed formulations DOSPA/DOPE (3/1) and DOTMA/DOPE (1/ 1), respectively. 400,401 The zwitterionic helper lipid DOPE is added to the formulations to enhance TE by direct fusion with the cell-membrane and bypassing cellular endocytosis, thereby avoiding inefficient endosomal escape. 402 Addition of cholesterol changes at certain concentrations the order of phospho-lipid assembly and results in more rigid liposomes. 403 However, when pure Chol is applied, it increases infectivity of Ad by forming Chol-Ad clusters via hydrophobic interactions with capsid proteins. 404,405 Formation and release. Complexes of lipids and VP are typically obtained by simple mixing of the VPs in aqueous solution with preformed liposomes on a thin film and subsequent incubation. 406,407 However, concerns regarding this method have been raised due to toxic and transduction ineffective aggregate formation. 408 Alternative encapsulation routes such as spontaneous self-assembly around VPs (Fig. 15A) have been demonstrated to circumvent these problems with partial success. 53,409,410 In general, it is assumed that TE of VPs is increased by accelerated cell entry due to the small size and positive charge of liposomal carriers 53,410 and further promoted by the socalled proton sponge effect, which has recently been critically   reviewed. 195 The proton sponge effect is caused by an increase of cationic charge at endosomal acidic pH, leading to osmotic swelling and accelerated liposome dissociation in the cytoplasm. 129,411 Apart from this, various supporting strategies have been developed to facilitate the endosomal escape by creating stimuli-responsive liposomes, for example, by employing pH-triggered release of Ad from DOPE/CHEMS 412 or MMPcleavable enzyme-responsive liposomes (Fig. 15B). 413 Most studies were conducted with complexes from cationic liposomes and VPs. Enhanced TE, shielding from immune response and broadened tropism of RV, 400,401,410,414 AAV, 415 Ad, 124,416,417 HSV, 418,419 as well as recently for oncolytic applications with reovirus 420 and alphavirus 421 were reported. Moreover, liposome vesicles from zwitterionic, cationic and PEG-lipid formulations can act as an artificial envelope for non-enveloped viruses and thereby enhance targeted delivery and prolong blood circulation lifetime (Fig. 15C and  D). 53,412,422 Further co-envelopment of Ad and siRNA yielded a dual active hybrid vector for multiple gene delivery. 423 Not all applications with lipids for virus delivery require preformed VP-lipid complexes. Parsons et al. applied lysolecithins as surfactants on host cells prior to LV administration for cystic fibrosis treatment in mice. In this way, a more effective, less immunogenic and persistent transduction was achieved already with a single injection. [424][425][426][427] Maguire et al.
were the first to report and isolate AAV in extracellular vesicles, which were released from AAV producer cells during the reproduction process. 428 These naturally encapsulated AAV showed enhanced TE and high biocompatibility at various application sites in vivo and has been reviewed elsewhere. 429 Modified lipids. One drawback of cationic liposomes in systemic administrations is unwanted interaction with serum proteins and subsequent clearance. 430 To tackle this issue, lipids have been covalently attached to PEG to achieve stealthy carriers with longer circulation times and protection from blood plasma protein adsorption. 166,431 These systems also enable tumor 413,432-434 and cell targeting. 435 In a different approach, PEG spacers were used as surface functionalization tools for liposomes, e.g. for covalent attachment of the CPP Tat or Pen. 391,436 Anionic lipids. Anionic liposomes, on the other hand, were introduced in the last decade and emerged as carriers for VVs with enhanced TE and low toxicity and immunogenicity. 123,437 They are more compatible with the abundance of negatively charged molecules in a physiological environment and therefore less prone to recognition by neutralizing antibodies. Due to the same charge polarity of VPs and anionic liposomes calcium-ions forming bridged complexes were required for encapsulation. 123,437 Surprisingly, enhanced TE is possible despite the anionic charge 438 even in a CAR-independent manner for encapsulated Ad. 123,422,437 The mechanism that promotes interactions of anionic liposomes with plasma membranes is still not fully understood. Possible reasons, such as longer interaction time due to reduced clearance and toxicity, 437,439,440 faster release of cargo, 441,442 or receptor mediated mechanisms 443 have been discussed. A thorough understanding of mechanisms promoting interactions of anionic liposomes with VPs is yet to be achieved. 444,445 This is of significant importance, especially with regard to oncolytic viral gene delivery, where an elevated presence of anionic lipids is observed on cancer cell membrane surface. 446,447 Nanoparticles Nanoparticles (NPs) are nanoscale materials, which have attracted growing interest in recent years due to their versatility and unique size-scaling properties. For example, NPs made from noble metals such as gold display strong surface plasmon absorption, making them an interesting material for local phototherapy and as an imaging agent in vivo. 448,449 Among manifold preparation techniques to obtain uniform, functional NPs, template-based strategies are well established. VPs are attractive templates due to the uniform, highly precise 3D structure of the virus capsid which are decorated with chemical functional groups. 450 NPs have also emerged in recent years as an invaluable asset for viral gene delivery. In general three approaches can be distinguished: (1) NPs and VP administered together without specific association between them; (2) VPs and NPs that are covalently bound, e.g. in a conjugate; and (3) noncovalent interactions such as electrostatic binding between VPs and NPs. Depending on the NP-VP size ratio, various architectures can be achieved. If the NPs size exceed the VPs size e.g. iron oxide NPs combined with AAV 120 VPs are decorated on the outer sphere of NPs. However, if the VPs are larger than NPs typically, NPs and VPs are combined either by coating VP surface via incubation and precipitation or by covalent attachment through functional groups on VP surface. For example, mineral shells for VPs can be created by precipitation from a solution of an inorganic salt and exhibit new biological and physical properties impacting stability, host cell adsorption and entry mechanism. 451 Covalent attachment of NPs to VPs is accessible by abundant lysine residues on capsid proteins and by genetically engineered functional groups, e.g. thiol-or azide-moieties.
Frequently applied NPs for viral gene delivery are made from calcium phosphate, gold, silica, graphene or iron oxide, are discussed in the following sections and summarized in Table 6.
Calcium phosphate. Calcium phosphate (Ca 3 (PO 4 ) 2 ) is highly abundant in human bone and teeth and one of the most important biominerals in Nature. 452 As a common material in humans and animals, Ca 3 (PO 4 ) 2 has a significant role also for viral infectivity, as reported for avian sarcoma virus. It is suggested that this virus occurs in aves in mineralized state and promotes transmission and bypassing of the zoonotic barrier to humans by a highly effective adsorption and Ca 3 (PO 4 ) 2 mediated internalization pathway into host cells. 113 At neutral conditions, Ca 3 (PO 4 ) 2 forms a solid precipitate around VPs, which can be degraded under acidic condition present in the endosome. 453 Ca 3 (PO 4 ) 2 is an ideal candidate as a biomineral due to its biocompatible properties and degradability into non-toxic ions after cellular entry (Fig. 16) has been applied as a virus shell by biomineralization and enabled enhanced CAR-independent infectivity, expanded tropism and shielding from neutralizing antibodies. 112,113 Chen et al. described a combinatorial design in which oncolytic Ad was subsequently coated with Ca 3 (PO 4 ) 2 , DOPA lipids and PEG to form a spherical three layered charged shell with almost neutral charge. This design masked the viral surface and lowered unwanted immune response and liver toxicity while improving TE to tumor tissue. 434 Similar, easy to fabricate virus-inorganic complexes from Ca 3 (PO 4 ) 2 are promising for systemic administration due to low administration dosage and prolonged circulation lifetime. 112 Recently, Huang et al.
proposed a more elaborate calcium-based shell for oncolytic Ad delivery, which was created from calcium and manganese carbonates. This shell prolonged in vivo circulation of VPs, protected them from immune response, significantly reduced their liver accumulation while still resulting in high tumor accumulation. In the acidic tumor microenvironment, Mn 2+ions were released and converted endogeneous H 2 O 2 into O 2 . Thereby oncolytic Ad duplication activity and antitumor effects were enhanced. Due to the increase of O 2 concentration, real time, label-free monitoring with magnetic resonance imaging was possible. 454 Inorganic particles from Ca 3 (PO 4 ) 2 can provide stealth biomineral shells by spontaneous coating of VPs surface and thereby have an influence on tropism, TE and biocompatibility. Combinations with other inorganic particles enable precise tuning of the release properties and give access to tailored carriers.
Gold. Gold NPs have been explored for various biomedical applications like diagnostics, radiotherapy or gene delivery due to their easy fabrication, biocompatibility, and functionalization. 455 Gold NPs were applied as covalent conjugates with VPs and reported either to retain or enhance TE 456,457 or attenuate infectivity, [458][459][460][461] depending on the capsid modification. Covalent conjugates were mainly achieved by attachment to the abundant lysine residues of capsid proteins. However,  modifications with high gold amounts interfered with capsid protein functions, which are important for virus infectivity. 456 Nevertheless, enhanced transduction was shown with various positively charged PEGylated Gold NPs functionalized with RGD epitope or azide group and coincubated with Ad, which formed spherical particles and exhibit higher TE to hMSCs compared to PEI or Lipofectamine coated Ad (Fig. 17). 457 A further interesting clinical application of Gold NPs is photothermal therapy, 462 which has been efficiently addressed in a combinatorial therapeutic approach with VPs. 456 Oncolytic Ad applied in combination with gold nanorods exhibit hyperthermia-induced enhancement of receptor-mediated endocytosis and TE in vivo, enabling locally induced tumor cell lysis. 463 Silica. Silica NPs are biodegradable minerals which are typically fabricated in a sol-gel process starting from tetramethyl orthosilicate or tetraethyl orthosilicate and are broadly employed as food additives or in biomedical applications. 464,465 Silica coatings for VPs are promising candidates to broaden the tropism of viral gene delivery, since a size dependent uptake by tumor cells is known for silica nanoparticles. 466 Recently, Ad coated with silica showed enhanced TE in glioma while reducing liver accumulation and immunogenicity. 125 Other formulations of silica have also been tested. Silica-based gels are synthesized through a simple sol-gel process, resulting in highly porous materials, suitable as implantable viral depots with long-term release. Typically, the degradation of silica gels is in the range of weeks and depends on the amount of water which is present. For example oncolytic Ad were encorporated in a biodegradable high water content silica-based gel and spatiotemporal release of oncolytic Ad in implanted tumor tissue site was achieved by gradually degradation in vivo. The virus could be stored in the silica gels and infectivity was preserved for a period of at least two years in vitro at 4°C. 128 Graphene. Graphene-based NPs display unique physiochemical properties, like electrical conductivity or photostability and have emerged as an abundant and low-cost resource for nanomedical application in recent years. 467 For example, they have been applied in tumor treatment, 468 photothermal therapeutic approaches, [469][470][471] virus detection 472 and have been used as drug 473,474 and gene delivery agents.
Since pioneering works from Kostarelos et al. 475,476 research efforts have been focussed on graphene as non-viral transfection agents, often in combination with grafted cationic polymers as adhesion promoters. [477][478][479][480] Recently, graphene have started to attract interest as viral delivery platforms. Graphene oxide (GO) sheets decorated with PEG-folate were shown to target susceptible cancer cells. 468 Based on these results, oncolytic measles virus was encapsulated by GO sheets functionalized with PEI and PEG-folate (Fig. 18). The encapsulated measles virus was protected against neutralizing antibodies, showed high tumor accumulation in vivo, exhibited higher infectivity and TE to cancer cells than virus alone and folate-free GO-PEI-PEG sheets. However, the release mechanism and in vivo stability of the VP-GO sheet cluster remain unexplored. 481 Another carbon-based material, carbon dots, spherical particles of few nanometer-size diameter, have been applied for viral gene delivery for the first time. 482 This relatively new material class, mostly derived from graphene, has been reported to show size-, charge-, and aggregation-dependent toxicity. 483 In order to stimulate cartilage repair, rAAV coding for the highly chondroreparative SOX9 transcription factor was complexed with various functionalized carbon dots, which resulted in significant increase of transduction to hMSCs with high cell viability. Especially carbon dots functionalized with 2-citric acid, poly(ethylene glycol) monomethyl ether and N,Ndimethylethylenediamine displayed efficient release and TE of rAAV. 482 Iron oxide. Iron oxide NPs have an outstanding position among inorganic NPs for viral gene delivery and have been intensively studied as an interface technique between physical and chemical delivery. They have especially attracted interest as the main component for remotely guided magnetic delivery vehicles. Gene delivery by using a gradient magnetic field, also   called magnetofection, has been reported to enhance TE of non-viral vectors and VVs after employing external magnetic fields for in vitro and in vivo applications [484][485][486] and have been successfully commercialized for example as AdenoMag (OZ Biosciences), 487 fluidMAG-Heparin (Chemicell) 120 or Endorem (Guerbet). 488 Magnetite (Fe 3 O 4 ) as superparamagnetic iron oxide nanoparticles (SPIONs) have been reported in several studies as a vehicle to enhance gene delivery efficiencies, e.g. for delivery of AAV, 115,489 Ad, 487,490 oncolytic Ad, 491,492 LV,484,493 and measles virus. 494 They are biocompatible and degradable and allow surface modification with various functionalities. 495,496 Coating SPIONs with biopolymers like heparin 120 or chitosan 497 can further augment TE with lower vector dosage and enable faster transduction (Fig. 19A). Moreover, polymeric coatings such as PEI or PEG on SPIONs as well as liposome encapsulation are reported to be highly beneficial for improving TE of Ad with reduced dose (Fig. 19B). 484,492,498 For example, PEI coatings give access to positively charged particles boosting cell interaction and endosomal escape by their proton sponge effect without affecting the superparamagnetic properties. 499 In a comparative study PEI, polybrene, or silica-PEI were coated onto iron oxide NPs and resulted in improved potency of oncolytic Ad. The highest antitumor activity in vivo was observed for silica-PEI coated iron oxide NPs, which was attributed to a higher shielding ability against neutralizing antibodies and higher magnetophoretic mobility. 491 In another study, various commercially available SPIONs were compared to each other in terms of susceptibility for aggregation and TE of attached alphavirus. Positively-charged NPs displayed rapid and effective isolation and concentration of VPs, resulting in higher TE than negatively charged NPs. 500 In general, VPs can be coated with modified SPIONs through simple incubation due to electrostatic interactions to obtain hybrid particles. 487,492 Beside this, covalent attachment of VPs onto SPIONs is regularly achieved by EDC-NHS chemistry coupling carboxy-functionalized SPIONs to the lysine residues on viral capsid. 489,498 Another, more laborious route to attach VPs onto SPIONs surfaces can be achieved by using a chelating approach, with a linker unit consisting of biotin-functionalized nickel ions conjugated to streptavidin modified SPIONs, which further bind to hexahistidine displaying AAV particles (Fig. 19C). This hybrid particle can transduce into otherwise non-permissive hNSCs by employing magnetic force. 115 Recently, precise manual virus stamping to single cells in culture as well as to mouse brain was reported by Schubert et al. This technique employs VPs covalently bound to silica-coated SPIONs which are loaded in a micropipette tip and brought into contact with the desired cells by a magnetic field (Fig. 19D). This method enables the delivery of multiple virus types to a single cell for multiple virus engineering or to track genetically modified single cells. 501 An interesting example to implement magnetofection with phototherapeutic tumor treatment was shown for AAV encoding a photosensitive apoptosis-inducing protein. AAV conju-   (Fig. 19E). This enzymatically-controlled release enabled targeted tumor cell lysis with an combinatorial approach of functional materials. 503 SPIONs are approved as contrast agents for magnetic resonance imaging, 504 which makes them a clinically promising vehicle for targeted delivery of VPs. One of main advantages of using magnetic NPs for VP delivery is the reduced vector dosage and short exposure times for efficient transduction due to controlled colocalization of cells and VPs. Magnetic NPs enable to deliver VPs to otherwise non-permissible cells and may enable the crossing of biophysical barriers in vivo in the future.

Small molecules
Small molecules are defined as organic compounds with a molecular mass below 900 Da and a physical size below 1 nm in pharmaceutical research. Today, the majority of drugs used as medicine in everyday life are small molecules, which are characterized by their high bioavailability and effective mode of action at the target site. 81 Research on small molecules that promote viral gene delivery has become more wide-spread in recent years.
The aim of adding small molecules during a transduction procedure is to achieve satisfactory TE with low vector doses to minimize side-effects and to enable transduction to cell types that are difficult to infect. The underlying mode of action of small molecules to achieve this aim is fundamentally different to previously discussed materials, which were interacting with VPs, forming a complex, aggregate or conjugate which in turn facilitates the interaction with cells. Here, small molecules interfere with multiple cellular processes, for example, by affecting gene transcription, cell entry mechanisms or DNA replication and thereby facilitating viral gene expression. The focus in small molecule research for viral gene delivery has been on dual mode drugs, which are promising due to their therapeutic as well as transduction enhancing properties. These molecules are usually simply added to VPs for application, even though covalent conjugation to VPs were also shown exemplarily. 506 In the following sections important findings are highlighted and summarized in Table 7 and Chart 3.
In early works, Miller et al. showed that DNA-damaging agents like UV-irradiation or cisplatin increased TE of AAV to non-dividing cells. 507 Later they introduced less cytotoxic DNAsynthesis inhibitors like aphidicolin or hydroxyurea and topoi-somerase inhibitors such as etoposide or camptothecin as enhancers of AAV transduction to fibroblasts. 508 Groschel et al. demonstrated that these cytostatic agents etoposide, camptothecin, taxol, and aphidicolin, interfere with various mechanisms of cell cycle progression and arrest the cell cycle at G 2 /M phase, which is a checkpoint to control DNA replication before nuclei division. Arresting the cell cycle in this phase boosted the early step of HIV virus replication and thereby enhanced TE. 135 Another well-known chemotherapeutic drug, doxorubicin, greatly enhanced rAAV infection to airway cells as well as neuronal cells with only mild cytotoxic effects at tested concentrations. Doxorubicin promoted rAAV accumulation at the nucleus, probably through proteasome inhibiting effects resulting in accelerated intracellular viral nuclear transport, but without affecting cell entry mechanisms. 509,510 The exact mechanism remained unclear and it was suggested that enhanced TE is more likely resulting from stimulated capsid uncoating and increased reverse transcription of viral genome than by increased cell entry. 135 Further cytokine stimulated downregulation of proteasome activity is believed to enhance TE. 511 Evidence for this hypothesis was found by addition of cytokines or proteasome inhibitors, particularly FDA approved bortezomib, resulting in enhanced TE of even low dosed LV to stem cells. [511][512][513] Furthermore, the proteasome inhibitor MG132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal) enhanced TE of rAAV to airway epithelia cells by modulating the ubiquitin-proteasome system in the endosomal environment. 514 Moreover, adenosine 5′-triphosphate-binding cassette transporter proteins, which efflux chemotherapeutic drugs and HIV protease inhibitors, play a significant role in reducing TE of LV into CD34 + stem cells. Enhanced TE of up to sixfold was observed after the addition of Verapamil, an adenosine 5′-triphosphate-binding cassetteinhibitor. 515 However, the identified chemotherapeutic compounds have huge disadvantages including damaging DNA and are therefore not ideal candidates for clinical use to healthy cells. In order to identify additional transduction enhancing drugs, high-throughput screens were conducted.
The first library screening for compounds promoting LV transduction identified 30 potential enhancers of viral transduction from 1280 investigated pharmaceutically active drugs. The non-DNA damaging, protein kinase activator phorbol 12-myristate 13-acetate (PMA) was found as a very promising candidate to enhance TE of LV into hard to transduce stem cells. Similar to previously discovered agents, PMA displayed interference with the cell cycle and inhibition of proliferation, suggesting that the transduction efficiency is influenced by processes within the cell rather than by promoted cell entry. 516 Another high-throughput screening with focus on enhancing TE of rAAV to HeLa cells investigated 2396 pharmaceutically known, mostly FDA approved compounds from which 13 compounds were identified as capable potentiators. In this screening, five main enhancer groups were identified: epipodophyllotoxins, inducers of DNA damage, effectors of epigenetic modification, anthracyclines, and proteasome inhibitors.

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The highest level of transduction, with approx. 50-100 times compared with virus-only transduction, was observed for the chemotherapeutic epipodophyllotoxin teniposide. It was observed that the TE varied significantly for the tested substances depending on cell type, which indicates an underlying correlation between mode-of-action and cell type. In unison with previous studies, the authors suggest that rAAV can be enhanced by two pathways, whose detailed mechanisms remain unknown: the inhibition of proteasomes and the induction of DNA damage. 517 A further screening for enhancing interferon sensitized oncolytic virus transduction to resistant cancer cells identified pyrrole-based molecules as potent enhancers. This was also feasible with AAV and Ad. High activities up to thousand-fold enhancement of viral transduction, tolerability and plasma stability were found for systemic administration in mice. The mode of action of these molecules was attributed to an inhibition of interferon-β production, which gives antiviral properties to resistant cancer cells. 518,519 In recent years, further pharmaceutically applied drugs were identified as promising viral gene delivery agents.
Prostaglandine E 2 enhanced LV transduction to HSCs by acting during the endocytosis phase via a yet unclear mechanism. [520][521][522] Lee et al. reported synergistic effects in terms of inducing apoptosis to malignant melanoma cells when AAV2 encoding a pro-apoptotic protein and the phytoactive compound 6-gingerol were combined. 6-Gingerol was previously reported to be effective against cancer and for the inhibition of angiogenesis.
The cytostatic activity has its source in the control of the cell cycle and subsequently inducing apoptosis. The authors proposed that AAV2 can bypass its usual endocytic receptormediated pathway in the presence of 6-gingerol and thereby enhancing apoptosis in melanoma cells more efficiently. 58 Furthermore, hydroxychloroquine, an approved antimalaria drug, has been applied for promoting AAV transduction in murine and human tissue in vitro and in vivo, e.g. in the retina with no adverse effects. Among the multimodal effects this drug might have on TE, one mode of action was proposed as an inhibition of TLR9 gene expression, which is a known sensor for anti-viral response of cells. 523 Intervention in cell regulation processes was similarly shown for Ad infection which was enantioselectively promoted by the bromodomain inhibitor (+)-JQ1. This molecule blocks acetylated lysine interaction with the bromodomain family proteins, which play a significant role in cell regulation and gene transcription. In this way, Ad infectivity and gene expression were elevated by suppression of regulating bromodomains, whose mechanisms still need to be elucidated. 524 Embelin, used as an adjuvant in oncolytic virotherapy was recently reported by Wang et al. Lymphoma cell lysis was induced by mitigating antiviral immunity against oncolytic virus upon addition of Embelin. The authors proposed that the infiltration of the oncolytic virus was facilitated by the disruption of Interleukin-6/STAT3 signalling and further promoted by the inherent ability of Embelin to induce apoptotic death to tumor cells. 525 An interesting approach was reported by Gong et al. who enhanced TE by increasing low-density lipoprotein-receptors expression with Rosuvastatin. Vesicular stomatitis virus was able to efficiently target and transduce in natural killer cells after addition of Rosuvastatin. Naturally, untreated killer cells lack of sufficient low-density lipoprotein-receptors, which are necessary for virus interaction. Statins, especially Rosuvastatin, can induce low-density lipoprotein expression and thereby facilitate virus-cell interaction. Additionally, geranylgeranylpyrophosphate suppresses cytotoxic effects of statins, resulting in a highly effective and powerful tool to enable genetic modification of natural killer cells. Genetically modified natural killer cells can, for example, express tumor antigen-specific receptors, which may pave the way for targeted treatment of malignant cells. 526 Small molecules, which are beneficial for disease treatment, eliciting host immune response while enabling efficient transduction are actively being pursued for clinical application. Pharmaceutically active drugs that have been found so far, are paving the way especially for oncolytic gene therapy and can be included easily in ex vivo gene transfer protocols. However, further research is indispensable for investigation of unwanted side effects of drugs combined with viruses during delivery and should be carefully evaluated in therapeutic application.

From development to usage
Most of the introduced materials for promoting viral gene transfer have not yet been translated to the clinic and are often not commercially available. Here, we will highlight materials that are emerging from the development stage to actual clini-   491,501,505 and small molecules. 517,518,523 Commercially available kits make transduction enhancers more broadly available and are thus attractive tools for use in research. For instance, several studies about CAR T-cell manufacturing employed kits of the peptidic enhancers Vectofusin®-1 (Miltenyi Biotec) and Protransduzin™ (JPT Peptide Technologies). 353,354,528,529 However, popular materials in research cannot seamlessly be transferred to the clinical context. Low cell viability in high concentrations limits the application of Polybrene® (Abbott Laboratories Corp.) in drug formulations. 530 More efficient enhancers than polybrene 531 are traded as jetPEI® (Polyplus transfection) and Lipofectamine™ (Thermo Fisher Scientific) and can be applied to a broad range of VVs. Kits for specific viruses are available, but their exact composition is a trade secret, e.g.: for LVs (ViraDuctin™ (Cell Biolabs), TransDux™ (System Biosciences), ViralPlus (Applied Biological Materials Inc.), SureENTRY (Qiagen)), as well as for Ad in the form of AdenoBoost™ (Sirion Biotech). High transduction rates with low-titer viruses are enabled with polycationic magnetic beads, commercialized as MACSductin™ (Miltenyi Biotec), ViroMag and AdenoMag (OZ Biosciences), Lenti-X™ Accelerator (Takara Bio) and MISSION® ExpressMag® (Sigma-Aldrich). In recent years, a growing number of such kits were made available in GMP grade addressing the demand for systematic investigation of transduction agents in a clinical context. Clinical trials use RetroNectin® (Takara Bio) in particular as the gold standard for ex vivo transduction of lenti-and retroviruses, even though a high multiplicity of infection and a time consuming coating protocol is needed. 361,[532][533][534] Poloxamers such as LentiBoost™ (Sirion Biotech) 535 are commonly used in formulations for in vivo clinical trials as enhancers and to prevent uncontrolled binding of VVs to injection devices. [536][537][538][539] While the majority of viral gene therapy drugs approved up until 2017 do not use additional transduction enhancers in their formulations, those approved since then have made use of agents such as protamine sulfate (ex vivo, Zynteglo), 540,541 Poloxamer 188 (in vivo, Zolgensma and Luxturna), 542,543 and human serum albumin (ex vivo, Yescarta, Kymriah). 544,545 Conclusions and outlook Viral gene delivery has come a long way and is now becoming increasingly therapeutically relevant. The rising number of approvals for clinical use in recent years holds a great promise for treatment of currently uncurable diseases. However, the full potential of gene therapy cannot be realized until safe and efficient delivery systems are available. In this review, we highlighted the most important approaches for viral gene delivery from a materials perspective. In particular, the high biocompatibility of peptidic delivery systems, and the superior shielding effects of polymer coatings and lipid encapsulation need to be emphasized. Both hydrogels and magnetic NPs allow spatially defined virus release, often in a time-controlled manner. Especially the local targeted delivery of VVs via magnetic nanoparticles to tumor sites has enormous potential in oncolytic virotherapy. The performance of delivery systems is determined by many experimental parameters, e.g. virus type, cell type, concentration, and incubation time. However, detailed large-scale comparative studies of different delivery agents are still missing. Therefore, it is often difficult or even impossible to draw conclusions on the relative performance of the delivery agents. In current viral gene delivery systems, immunogenicity, native tropism, unspecific delivery, and poor efficiency at low vector doses are remaining hurdles to be overcome. The materials introduced here have the potential to establish hybrid VVs for clinical applications. However, clinical data of administration to humans remains scarce.
Gene delivery as a therapeutic approach is one of the major medicinal and biotechnological advances of this century. We expect that novel bioengineered viruses promoted by synthetic materials are destined for future efficient and safe clinical applications.

Conflicts of interest
There are no conflicts to declare.